Inhibitors of ret

ABSTRACT

Described herein are compounds that inhibit wild-type RET and its resistant mutants, pharmaceutical compositions including such compounds, and methods of using such compounds and compositions.

CLAIM OF PRIORITY

This application claims priority from U.S. Ser. No. 62/309,748, filed Mar. 17, 2016, which is incorporated herein in its entirety.

This invention relates to inhibitors of RET that are active against wild-type RET and its resistant mutants.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 15, 2017, is named B2094-7022WO_SL.txt and is 32,316 bytes in size.

BACKGROUND

RET (rearranged during transfection) is a receptor tyrosine kinase that activates multiple downstream pathways involved in cell proliferation and survival. RET fusions are implicated in several cancers including papillary thyroid carcinoma and non-small cell lung cancer. A genomics analysis on the landscape of kinase fusions identified RET fusions in breast and colon cancer patient samples, providing therapeutic rationale for the use of RET inhibitors in multiple patient subpopulations.

The identification of RET fusions as drivers in some cancers prompted the use of approved multi-kinase inhibitors with RET inhibitory activity to treat patients whose tumors express a RET fusion protein. However, these drugs cannot always be dosed at the levels required to sufficiently inhibit RET due to toxicities that result from inhibition of targets other than RET. Further, one of the greatest challenges in treating cancer is the ability of tumor cells to become resistant to therapy. Kinase reactivation via mutation is a common mechanism of resistance. When resistance occurs, the patient's treatment options are often very limited, and the cancer progresses, unchecked, in most instances. There is thus a need for compounds that inhibit RET, as well as its resistant mutants.

SUMMARY

The present invention provides inhibitors of RET and RET mutants, e.g., RET resistant mutants (as defined herein), for example, inhibitors of structural Formula (I) and pharmaceutically acceptable salts and compositions thereof. The present invention further provides methods of using the compounds of the invention, and pharmaceutically acceptable salts and compositions thereof, to inhibit the activity of RET or RET mutants in a cell or patient. The present invention still further provides methods for using the compounds of the invention, and pharmaceutically acceptable salts and compositions thereof, to treat a subject suffering from a condition mediated by aberrant RET activity, e.g., cancer.

In one aspect, the invention features a compound of structural Formula (I) or a pharmaceutically acceptable salt thereof:

wherein each of ring A, X¹, X², R¹, R², R^(3a), R^(3b), R⁴, R⁵, R⁶, R⁷, R^(8a), R^(8b), R⁹, m and n is defined as described herein.

In another aspect, the present invention provides pharmaceutical compositions comprising a compound of structural Formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a method for inhibiting RET activity in a cell or in a patient. In some embodiments, said method comprises the step of contacting the cell or administering to the patient a compound of structural Formula (I) or a pharmaceutically acceptable salt or composition thereof. In some embodiments, the cell or patient has, or has been identified as having, a RET alteration, e.g., a RET mutation, e.g., a fusion or point mutation. In some embodiments, the patient comprises a RET-altered cell, cancer, gene, or gene product.

In another aspect, the present invention provides a method for treating a subject suffering from a condition mediated by aberrant RET activity. In some embodiments, said method comprises administering to the subject a therapeutically effective amount of a compound of structural Formula (I) or a pharmaceutically acceptable salt or composition thereof. In some embodiments, the subject has or has been identified as having (e.g., wherein a cancer cell in the subject has or has been identified as having) a RET alteration, e.g., a RET mutation, e.g., a fusion or point mutation. In some embodiments, a cell, cancer, gene, or gene product from the subject is or has been identified as being RET-altered. In some embodiments, the subject has or has been identified as having (e.g., a cancer cell in the subject has or has been identified as having) a RET alteration, e.g., a RET mutation, e.g., a fusion or point mutation.

In some embodiments, the condition mediated by aberrant RET activity is a condition mediated by any RET activity that is not normal e.g., any activity due to a RET-altered gene or gene product, which affects the amount or activity of the gene or gene product as compared to the normal or wild-type gene. In embodiments, the condition mediated by aberrant RET activity is a familial or sporadic cancer, e.g., a solid tumor such as thyroid, lung, breast, or pancreatic. In embodiments, the condition mediated by aberrant RET activity is irritable bowel syndrome (IBS). In embodiments, the aberrant RET activity promotes the condition, such that inhibition of RET ameliorates at least one symptom of the condition. In embodiments, the aberrant RET activity comprises increased RET activity or expression level, gain of function mutation, and/or constitutive activation of RET. In embodiments, the aberrant RET activity corresponds to aberrant amounts of RET, e.g., aberrant nucleic acid or protein amounts.

In another aspect, the present invention provides a method for treating a subject who has developed resistance to a cancer treatment. In some embodiments, said method comprises administering to the subject a therapeutically effective amount of a compound of structural Formula (I) or a pharmaceutically acceptable salt or composition thereof. In some embodiments, the subject has developed resistance to a wild-type RET inhibitor. In embodiments, the cancer treatment to which the subject is resistant is a wild-type RET inhibitor that is active against the wild-type RET, but less active, e.g., much less active, against one or more mutated forms of RET. In some embodiments, the wild-type RET inhibitor is selected from ponatinib, carbozanitib, and vandetanib.

In another aspect, the present invention provides a use of a compound or pharmaceutical composition described herein, e.g., a compound of structural Formula I, (Ia), (Ib), II, (IIa), or (IIb) described herein (e.g., a compound in Table 1) or a pharmaceutically acceptable salt or composition thereof, in the manufacture of a medicament for inhibiting RET activity in a cell or in a subject. In embodiments, the cell or subject has, or has been identified as having, a RET alteration, e.g., a RET mutation, e.g., a fusion or point mutation. In embodiments, a cell, cancer, gene, or gene product from the subject is or has been identified as being RET-altered.

In another aspect, the present invention provides a use of a compound or pharmaceutical composition described herein, e.g., a compound of structural Formula I, (Ia), (Ib), II, (IIa), or (IIb) described herein (e.g., a compound in Table 1) or a pharmaceutically acceptable salt or composition thereof, in the manufacture of a medicament for treating a subject suffering from a condition mediated by aberrant RET activity. In embodiments, the subject has, or has been identified as having (e.g., wherein a cancer cell in the subject has, or has been identified as having) a RET alteration, e.g., a RET mutation, e.g., a fusion or point mutation. In embodiments, a cell, cancer, gene, or gene product from the subject is or has been identified as being RET-altered.

In another aspect, the present invention provides a use of a compound or pharmaceutical composition described herein, e.g., a compound of structural Formula I, (Ia), (Ib), II, (IIa), or (IIb) described herein (e.g., a compound in Table 1) or a pharmaceutically acceptable salt or composition thereof, in the manufacture of a medicament for treating a subject who has developed resistance to a cancer treatment.

In another aspect, the present invention provides a method of preventing development of one or more RET-altered cell, cancer, gene, or gene product, in a cell or in a patient, comprising the step of contacting the cell or administering to the patient a compound according to structural Formula (I).

In another aspect, the present invention provides a method of treating a subject suffering from cancer comprising the steps of:

-   -   a. receiving information related to a RET sequence, e.g.,         information related to a RET-altered gene or gene product, e.g.,         having a RET fusion or point mutation; and     -   b. administering to the subject a compound described herein         (e.g., a compound having a lower IC₅₀ for RET than for KDR, a         compound having a similar IC₅₀ for wild-type RET compared to         mutant RET, and/or a compound of structural Formula I, (Ia),         (Ib), II, (IIa), or (IIb) described herein (e.g., a compound in         Table 1)), or a pharmaceutical composition comprising the         compound, if the information indicates a RET-altered cell,         cancer, gene, or gene product.

In embodiments, the subject is administered a cancer treatment other than a compound of structural Formula (I) or (II) if the information indicates that the subject has a wild-type RET sequence. In embodiments, the cancer treatment is a wild-type RET inhibitor.

In another aspect, the present invention provides a method of treating cancer in a subject, said method comprising:

a. obtaining a biological sample (e.g., a tumor biopsy) from a human subject;

b. detecting whether a RET-altered cell, cancer, gene, or gene product, e.g., having a fusion or point mutation, is present in the biological sample;

c. identifying the subject as responsive to a compound described herein (e.g., a compound having a lower IC₅₀ for RET than for KDR, a compound having a similar IC₅₀ for wild-type RET compared to mutant RET, and/or a compound of structural Formula I, (Ia), (Ib), II, (IIa), or (IIb) described herein (e.g., a compound in Table 1)) when the presence of the RET-altered cell, cancer, gene, or gene product in the biological sample is detected; and

d. administering an effective amount of the compound to the subject.

In another aspect, the present invention provides a method of treating cancer in a subject, said method comprising:

-   -   a. determining if, having determined if, or receiving         information that the subject has a RET-altered cell, cancer,         gene, or gene product, e.g., having a fusion or point mutation;     -   b. identifying the subject as responsive to a compound described         herein (e.g., a compound having a lower IC₅₀ for RET than for         KDR, a compound having a similar IC₅₀ for wild-type RET compared         to mutant RET, and/or a compound of structural Formula I, (Ia),         (Ib), II, (IIa), or (IIb) described herein (e.g., a compound in         Table 1)) when the subject has a RET-altered cell, cancer, gene,         or gene product; and     -   c. administering an effective amount of the compound to the         subject.

In another aspect, the present invention provides a method of diagnosing cancer in a subject, said method comprising:

a. obtaining a biological sample (e.g., a tumor biopsy) from a human subject;

b. detecting whether a RET-altered cell, cancer, gene, or gene product, e.g., having a fusion or point mutation, is present in the biological sample;

c. diagnosing the subject with cancer when the presence of the RET-altered cell, cancer, gene, or gene product in the biological sample is detected.

In another aspect, the present invention provides a method of predicting the efficacy of a compound described herein (a compound having a lower IC₅₀ for RET than for KDR, a compound having a similar IC₅₀ for wild-type RET compared to mutant RET, and/or a compound of structural Formula I, (Ia), (Ib), II, (IIa), or (IIb) described herein (e.g., a compound in Table 1)) in a treatment of cancer in a subject comprising the step of:

determining if, having determined if, or receiving information that the subject has a RET-altered cell, cancer, gene, or gene product, e.g., having a mutation, e.g., a fusion or point mutation, e.g., by a method selected from hybridization-based methods, amplification-based methods, microarray analysis, flow cytometry analysis, DNA sequencing, next-generation sequencing (NGS), primer extension, PCR, in situ hybridization, dot blot, and Southern blot;

wherein said determining if, having determined if, or receiving information is predictive of efficacy of the compound in the treatment.

In some embodiments of any of the methods and uses herein, the method further comprises administering to the subject a compound described herein, e.g., (e.g., a compound having a lower IC₅₀ for RET than for KDR, a compound having a similar IC₅₀ for wild-type RET compared to mutant RET, and/or a compound of structural Formula I, (Ia), (Ib), II, (IIa), or (IIb) described herein (e.g., a compound in Table 1)), e.g., responsive to a determination or diagnosis made by a method described herein.

In some embodiments of any of the methods and uses herein, the RET-altered cell, cancer, gene, or gene product comprises a fusion mutation, e.g., CCDC6-RET or KIF5B-RET or a fusion of Table 3. In embodiments, the RET-altered cell, cancer, gene, or gene product comprises a point mutation of amino acid position 634, 918, or 804 of RET, or a point mutation at a position listed in Table 4, e.g., a point mutation specified in Table 4. In some embodiments, the RET-altered cell, cancer, gene, or gene product comprises a point mutation at amino acid 634, 918, or 804, 806, 810, 865, 870, 891, e.g., is selected from RET C634W, M918T, V804L, V804E, V804M, V804L, V806C, Y806C, Y806S, Y806N, Y806H, G810R, G810S, L865V, L870F, and S891A. In some embodiments, the RET alteration is located at least partially within, or is located wholly within one or more of: the N-terminal extracellular domain (e.g., within one or more cadherin-like repeats and/or the cysteine-rich region), the transmembrane domain, or the tyrosine kinase domain (e.g., within one or more of the ATP binding site or proton acceptor site). In some embodiments, the RET fusion further comprises a point mutation, e.g., KIF5B-RET (V804L) or KIF5B-RET (V804M).

In some embodiments of any of the methods and uses herein, the subject suffers from a cancer selected from colorectal cancer, lung cancer (e.g., a lung adenocarcinoma, e.g., NSCLC), thyroid cancer (e.g., medullary thyroid cancer), or leukemia. In embodiments, the subject suffers from a cancer listed herein, e.g., in Table 3.

In some embodiments, the cancer is wild-type RET.

In some embodiments, the cancer is lung cancer, e.g., a lung adenocarcinoma, and the RET-altered cell, cancer, gene, or gene product comprises a CCDC6-RET fusion. In some embodiments, the cancer is lung adenocarcinoma and the RET-altered cell, cancer, gene, or gene product comprises a KIF5B-RET fusion. In some embodiments, the cancer is lung adenocarcinoma and the RET-altered cell, cancer, gene, or gene product comprises KIF5B-RET (V804L). In some embodiments, the lung adenocarcinoma is NSCLC.

In some embodiments, the cancer is thyroid cancer, e.g., medullary thyroid cancer, and the RET-altered cell, cancer, gene, or gene product comprises a C634W mutation.

In some embodiments, the cancer is leukemia and the RET-altered cell, cancer, gene, or gene product comprises a KIF5B-RET fusion.

In some embodiments, the cancer is thyroid cancer and the RET-altered cell, cancer, gene, or gene product comprises a M918T mutation. In some embodiments, the cancer is medullary thyroid cancer and the RET-altered cell, cancer, gene, or gene product comprises a M918T mutation.

In some embodiments, the cancer is leukemia and the RET-altered cell, cancer, gene, or gene product comprises comprises KIF5B-RET (V804L) or KIF5B-RET (V804M) fusion.

In some embodiments, the cancer is thyroid cancer and the RET-altered cell, cancer, gene, or gene product comprises comprises a CCDC6-RET fusion. In some embodiments, the cancer is colorectal cancer and the RET-altered cell, cancer, gene, or gene product comprises CCDC6-RET fusion. In some embodiments, the cancer is colorectal cancer and the RET-altered cell, cancer, gene, or gene product comprises CCDC6-RET (V804M) fusion.

In some embodiments of any of the methods and uses herein, the compound or pharmaceutical composition has a lower IC₅₀ for RET than for KDR, e.g., has a KDR/RET IC₅₀ ratio of at least 3×, 4×, 5×, 10×, 20×, 50×, or 100×, and optionally up to 50× or 100×. In embodiments, the compound or pharmaceutical composition has a KDR/RET IC₅₀ ratio of between 3×-4×, 4×-5×, 5×-10×, 10×-20×, 20×-50×, or 50×-100×. In some embodiments, the compound or pharmaceutical composition has a similar IC₅₀ for wild-type RET compared to mutant RET (e.g., for V804L RET or V804E RET), e.g., has a wild-type/mutant IC₅₀ ratio of no more than 3×, 2×, 1.5×, 1×, or 0.5×, e.g., of between 3× and 0.5×.

In some embodiments of any of the methods and uses herein, the subject does not develop a RET-altered cell, cancer, gene, or gene product for at least 1, 2, 3, 6, 9, 12, 24, or 36 months after initiation of administration of the compound. In some embodiments, the compound is administered as a first line therapy. In some embodiments, the compound is administered to a treatment-naïve subject. In some embodiments, the compound is not administered in combination with another kinase inhibitor. In some embodiments, the compound is not administered in combination with another RET inhibitor. For example, the compound can be administered as a monotherapy or in combination with one or other agents which are not kinase inhibitors, e.g., not RET inhibitors.

In some embodiments of any of the methods and uses herein, the compound is administered at an amount sufficient to reach at least 70%, 80%, 90%, or 95% inhibition of RET in vivo.

EMBODIMENTS OF THE INVENTION Definitions

As used herein, the terms a “patient,” “subject,” “individual,” and “host” refer to either a human or a non-human animal suffering from or suspected of suffering from a disease or disorder associated with aberrant RET expression (i.e., increased RET activity caused by signaling through RET) or biological activity.

“Treat” and “treating” such a disease or disorder refers to ameliorating at least one symptom of the disease or disorder. These terms, when used in connection with a condition such as a cancer, refer to one or more of: impeding growth of the cancer, causing the cancer to shrink by weight or volume, extending the expected survival time of the patient, inhibiting tumor growth, reducing tumor mass, reducing size or number of metastatic lesions, inhibiting the development of new metastatic lesions, prolonging survival, prolonging progression-free survival, prolonging time to progression, and/or enhancing quality of life.

The term “therapeutic effect” refers to a beneficial local or systemic effect in animals, particularly mammals, and more particularly humans, caused by administration of a compound or composition of the invention. The phrase “therapeutically-effective amount” means that amount of a compound or composition of the invention that is effective to treat a disease or condition caused by over expression of RET or aberrant RET biological activity at a reasonable benefit/risk ratio. The therapeutically effective amount of such substance will vary depending upon the subject and disease or condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of skill in the art.

As used herein, “developing resistance” means that when a drug is first administered to the patient, the patient's symptoms improve, whether measured by decrease in tumor volume, a decrease in the number of new lesions, or some other means that a physician uses to judge disease progression; however, those symptoms stop improving, or even worsen at some point. At that time, the patient is said to have developed resistance to the drug.

“Alteration” as used herein, of a gene or gene product (e.g., the RET gene or gene product) refers to the presence of a mutation or mutations within the gene or gene product, e.g., a mutation, which of the gene or gene product, as compared to the normal or wild-type gene. The alteration can be in amount, structure, and/or activity in a cancer tissue or cancer cell, as compared to its amount, structure, and/or activity, in a normal or healthy tissue or cell (e.g., a control), and is associated with a disease or condition, such as cancer. For example, an alteration which is associated with cancer, or predictive of responsiveness to an anti-cancer therapeutic, can have an altered nucleotide sequence (e.g., a mutation), amino acid sequence, chromosomal translocation, intra-chromosomal inversion, copy number, expression level, protein level, protein activity, or methylation status, in a cancer tissue or cancer cell, as compared to a normal, healthy tissue or cell. Exemplary mutations include, but are not limited to, point mutations (e.g., silent, missense, or nonsense), deletions, insertions, inversions, linking mutations, duplications, translocations, inter- and intra-chromosomal rearrangements. Mutations can be present in the coding or non-coding region of the gene, e.g., a 3′ UTR or 5′ UTR.

A subject having “altered RET” refers to a subject comprising a RET alteration, e.g., in one or more of their cancer cells.

A “RET-altered” cell, cancer, gene, or gene product refers to a cell, cancer, gene, or gene product comprising a RET alteration as described herein.

“Aliphatic group” means a straight-chain, branched-chain, or cyclic hydrocarbon group and includes saturated and unsaturated groups, such as an alkyl group, an alkenyl group, and an alkynyl group.

“Alkylene” refers to a divalent radical of an alkyl group, e.g., —CH₂—, —CH₂CH₂—, and —CH₂CH₂CH₂—.

“Alkenyl” means an aliphatic group containing at least one double bond.

“Alkoxyl” or “alkoxy” means an alkyl group having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. The term “haloalkoxy” refers to an alkoxy in which one or more hydrogen atoms are replaced by halo, and includes alkoxy moieties in which all hydrogens have been replaced by halo (e.g., perfluoroalkoxy).

“Alkyl” refers to a monovalent radical of a saturated straight or branched hydrocarbon, such as a straight or branched group of 1-12, 1-10, or 1-6 carbon atoms, referred to herein as C₁-C₁₂ alkyl, C₁-C₁₀ alkyl, and C₁-C₆ alkyl, respectively. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, etc.

“Alkenylene” refers to an alkenyl group having two connecting points. For example, “ethenylene” represents the group —CH═CH—. Alkenylene groups can also be in an unsubstituted form or substituted form with one or more substituents.

“Alkynyl” refers to a straight or branched hydrocarbon chain containing 2-12 carbon atoms and characterized in having one or more triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propargyl, and 3-hexynyl. One of the triple bond carbons may optionally be the point of attachment of the alkynyl substituent.

“Alkynylene” refers to an alkynyl having two connecting points. For example, “ethynylene” represents the group —C≡C—. Alkynylene groups can also be in an unsubstituted form or substituted form with one or more substituents.

“Aromatic ring system” is art-recognized and refers to a monocyclic, bicyclic or polycyclic hydrocarbon ring system, wherein at least one ring is aromatic.

“Aryl” refers to a monovalent radical of an aromatic ring system. Representative aryl groups include fully aromatic ring systems, such as phenyl, naphthyl, and anthracenyl, and ring systems where an aromatic carbon ring is fused to one or more non-aromatic carbon rings, such as indanyl, phthalimidyl, naphthimidyl, or tetrahydronaphthyl, and the like.

“Arylene” refers to a divalent aryl, wherein “aryl” is as defined herein.

“Arylalkyl” or “aralkyl” refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of “arylalkyl” or “aralkyl” include benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups.

“Aryloxy” refers to —O-(aryl), wherein the aryl moiety is as defined herein.

“Halo” refers to a radical of any halogen, e.g., —F, —Cl, —Br, or —I.

“Heteroalkyl” refers to an optionally substituted alkyl, which has one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus or combinations thereof. A numerical range may be given, e.g. C₁-C₆ heteroalkyl which refers to the number of carbons in the chain, which in this example includes 1 to 6 carbon atoms. For example, a —CH₂OCH₂CH₃ radical is referred to as a “C₃” heteroalkyl. Connection to the rest of the molecule may be through either a heteroatom or a carbon in the heteroalkyl chain.

“Heteroalkylene” refers to a divalent optionally substituted alkyl, which has one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus or combinations thereof.

“Carbocyclic ring system” refers to a monocyclic, bicyclic or polycyclic hydrocarbon ring system, wherein each ring is either completely saturated or contains one or more units of unsaturation, but where no ring is aromatic.

“Carbocyclyl” refers to a monovalent radical of a carbocyclic ring system. Representative carbocyclyl groups include cycloalkyl groups (e.g., cyclobutyl, cyclopentyl, cyclohexyl and the like), and cycloalkenyl groups (e.g., cyclopentenyl, cyclohexenyl, cyclopentadienyl, and the like).

“Cycloalkyl” refers to a cyclic, bicyclic, tricyclic, or polycyclic non-aromatic hydrocarbon groups having 3 to 12 carbons. Any substitutable ring atom can be substituted (e.g., by one or more substituents). The cycloalkyl groups can contain fused or spiro rings. Fused rings are rings that share a common carbon atom. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclohexyl, methylcyclohexyl, adamantyl, and norbornyl.

“Cycloalkylalkyl” refers to a -(cycloalkyl)-alkyl radical where cycloalkyl and alkyl are as disclosed herein. The “cycloalkylalkyl” is bonded to the parent molecular structure through the cycloalkyl group.

“Heteroaromatic ring system” is art-recognized and refers to monocyclic, bicyclic or polycyclic ring system wherein at least one ring is both aromatic and comprises at least one heteroatom (e.g., N, O or S); and wherein no other rings are heterocyclyl (as defined below). In certain instances, a ring which is aromatic and comprises a heteroatom contains 1, 2, 3, or 4 ring heteroatoms in such ring.

“Heteroaryl” refers to a monovalent radical of a heteroaromatic ring system. Representative heteroaryl groups include ring systems where (i) each ring comprises a heteroatom and is aromatic, e.g., imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, thiophenyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl; (ii) each ring is aromatic or carbocyclyl, at least one aromatic ring comprises a heteroatom and at least one other ring is a hydrocarbon ring or e.g., indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, pyrido[2,3-b]-1,4-oxazin-3-(4H)-one, 5,6,7,8-tetrahydroquinolinyl and 5,6,7,8-tetrahydroisoquinolinyl; and (iii) each ring is aromatic or carbocyclyl, and at least one aromatic ring shares a bridgehead heteroatom with another aromatic ring, e.g., 4H-quinolizinyl.

“Heteroarylene” refers to a divalent heteroaryl, wherein “heteroaryl” is as defined herein.

“Heterocyclic ring system” refers to monocyclic, bicyclic and polycyclic ring systems where at least one ring is saturated or partially unsaturated (but not aromatic) and that ring comprises at least one heteroatom. A heterocyclic ring system can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted.

“Heterocyclyl” refers to a monovalent radical of a heterocyclic ring system. Representative heterocyclyls include ring systems in which (i) every ring is non-aromatic and at least one ring comprises a heteroatom, e.g., tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl; (ii) at least one ring is non-aromatic and comprises a heteroatom and at least one other ring is an aromatic carbon ring, e.g., 1,2,3,4-tetrahydroquinolinyl, 1,2,3,4-tetrahydroisoquinolinyl; and (iii) at least one ring is non-aromatic and comprises a heteroatom and at least one other ring is aromatic and comprises a heteroatom, e.g., 3,4-dihydro-1H-pyrano[4,3-c]pyridine, and 1,2,3,4-tetrahydro-2,6-naphthyridine.

“Heterocyclylalkyl” refers to an alkyl group substituted with a heterocyclyl group.

“Cyano” refers to a —CN radical.

“Nitro” refers to —NO₂.

“Hydroxy” or “hydroxyl” refers to —OH.

Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention. Thus, when a disclosed compound is named or depicted by a structure without specifying the stereochemistry and has one or more chiral centers, it is understood to represent all possible stereoisomers of the compound, as well as enantiomeric mixtures thereof. When a disclosed compound is named or depicted by a structure specifying stereochemistry at each chiral center, it is understood to represent only the compound having the designated stereochemistry at such chiral centers. However, when a disclosed compound specifies stereochemistry at some, but not all chiral centers, it is understood to represent all possible stereoisomers at the non-specified chiral centers of the compound, as well as enantiomeric mixtures thereof.

If, for instance, a particular enantiomer of compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.

The “enantiomeric excess” or “% enantiomeric excess” of a composition can be calculated using the equation shown below. In the example shown below a composition contains 90% of one enantiomer, e.g., the S enantiomer, and 10% of the other enantiomer, i.e., the R enantiomer.

ee=(90−10)/100=80%.

Thus, a composition containing 90% of one enantiomer and 10% of the other enantiomer is said to have an enantiomeric excess of 80%.

The compounds or compositions described herein may contain an enantiomeric excess of at least 50%, 75%, 90%, 95%, or 99% of one form of the compound, e.g., the S-enantiomer. In other words such compounds or compositions contain an enantiomeric excess of the S enantiomer over the R enantiomer.

The compounds described herein may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example deuterium (²H), tritium (³H), carbon-13 (¹³C), or carbon-14 (¹⁴C). All isotopic variations of the compounds disclosed herein, whether radioactive or not, are intended to be encompassed within the scope of the present invention. In addition, all tautomeric forms of the compounds described herein are intended to be within the scope of the invention.

The compound can be useful as the free base or as a salt. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19.)

As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted”, whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at each position. Combinations of substituents envisioned under this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable”, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

Suitable substituents for an optionally substituted alkyl, alkylene, heteroalkyl, heteroalkylene, carbocyclyl, heterocyclyl, aryl group and heteroaryl group include halogen, ═O, —CN, —OR^(c), —NR^(d)R^(e), —S(O)_(k)R^(c), —NR^(c)S(O)₂R^(c), —S(O)₂NR^(d)R^(e), —C(═O)OR^(c), —OC(═O)OR^(c), —OC(═O)R^(c), —OC(═S)OR^(c), —C(═S)OR^(c), —O(C═S)R^(c), —C(═O)NR^(d)R^(e), —NR^(c)C(═O)R^(c), —C(═S)NR^(d)R^(e), —NR^(c)C(═S)R^(c), —NR^(c)(C═O)OR^(c), —O(C═O)NR^(d)R^(e), —NR^(c)(C═S)OR^(c), —O(C═S)NR^(d)R^(e), —NR^(c)(C═O)NR^(d)R^(e), —NR^(c)(C═S)NR^(d)R^(e), —C(═S)R^(c), —C(═O)R^(c), C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl, carbocyclyl, (C₁-C₆-alkylene)-carbocyclyl, (C₁-C₆-heteroalkylene)-carbocyclyl, heterocyclyl, (C₁-C₆-alkylene)-heterocyclyl, (C₁-C₆-heteroalkylene)-heterocyclyl, aryl, (C₁-C₆-alkylene)-aryl, (C₁-C₆-heteroalkylene)-aryl, heteroaryl, (C₁-C₆-alkylene)-heteroaryl, or (C₁-C₆-heteroalkylene)-heteroaryl, wherein each of said alkyl, alkylene, heteroalkyl, heteroalkylene, carbocyclyl, heterocyclyl, aryl and heteroaryl are optionally substituted with one or more of halogen,

OR^(c), —NO₂, —CN, —NR^(c)C(═O)R^(c), —NR^(d)R^(e), —S(O)_(k)R^(c), —C(═O)OR^(c), —C(═O)NR^(d)R^(e), —C(═O)R^(c), C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆ heteroalkyl, and wherein R^(c) is hydrogen, hydroxy, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, carbocyclyl, (C₁-C₆-alkylene)-carbocyclyl, (C₁-C₆-heteroalkylene)-carbocyclyl, heterocyclyl, (C₁-C₆-alkylene)-heterocyclyl, (C₁-C₆-heteroalkylene)-heterocyclyl, aryl, (C₁-C₆-alkylene)-aryl, (C₁-C₆-heteroalkylene)-aryl, heteroaryl, (C₁-C₆-alkylene)-heteroaryl, or (C₁-C₆-heteroalkylene)-heteroaryl, each of which is optionally substituted with one or more of halogen, hydroxy, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ heteroalkyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl; R^(d) and R^(e) are each independently selected from hydrogen, C₁-C₆ alkyl, or C₁-C₆ heteroalkyl; and k is 0, 1 or 2. The invention is not intended to be limited in any manner by the above exemplary listing of substituents.

Compounds

In one aspect, the present invention features a compound having the structural Formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

ring A is an aryl or heteroaryl ring;

each of X¹ and X² is independently selected from N and C(R⁶);

each R¹ and each R⁷ is independently selected from selected from C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxy, halo, C₁-C₆ heteroalkyl, cycloalkyl, aryl, heteroaryl, aryloxy, aralkyl, heterocyclyl, heterocyclylalkyl, nitro, cyano, —C(O)R, —OC(O)R, —C(O)OR, —(C₁-C₆ alkylene)-C(O)R, —SR, —S(O)₂R, —S(O)₂—N(R)(R), —(C₁-C₆ alkylene)-S(O)₂R, —(C₁-C₆ alkylene)-S(O)₂—N(R)(R), —N(R)(R), —C(O)—N(R)(R), —N(R)—C(O)R, —N(R)—C(O)OR, —(C₁-C₆ alkylene)-N(R)—C(O)R, —N(R)S(O)₂R, and —P(O)(R)(R); wherein each of alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, cycloalkyl, aryl, heteroaryl, aryloxy, aralkyl, heterocyclyl, and heterocyclylalkyl is independently substituted with 0-5 occurrences of R^(a); or two R¹ or two R⁷ are taken together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclyl ring independently substituted with 0-5 occurrences of R^(b);

each of R², R^(3a), R^(3b), R⁴, R^(8a) and R^(8b) is independently selected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, halo, hydroxyl, C₁-C₆ heteroalkyl, and —N(R)(R); wherein each alkyl, alkoxy, and heteroalkyl is optionally and independently substituted with 0-5 occurrences of R^(a);

each of R⁵ and R⁹ is independently selected from hydrogen, C₁-C₆ alkyl, and C₁-C₆ heteroalkyl; wherein each alkyl and heteroalkyl is optionally and independently substituted with 0-5 occurrences of R^(a);

each R⁶ is independently selected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, halo, C₁-C₆ heteroalkyl, and —N(R)(R); wherein each alkyl, alkoxy, and heteroalkyl is optionally and independently substituted with 0-5 occurrences of R^(a);

each R is independently selected from hydrogen, hydroxyl, halo, thiol, C₁-C₆ alkyl, C₁-C₆ thioalkyl, C₁-C₆ alkoxy, C₁-C₆ heteroalkyl, cycloalkyl, cycloalkylalkyl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl, wherein each of alkyl, thioalkyl, alkoxy, heteroalkyl, cycloalkyl, cycloalkylalkyl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl is independently substituted with 0-5 occurrences of R^(a), or two R together with the atom(s) to which they are attached form a cycloalkyl or heterocyclyl ring independently substituted with 0-5 occurrences of R^(b);

each R^(a) and each R^(b) is independently selected from C₁-C₆ alkyl, halo, hydroxyl, C₁-C₆ heteroalkyl, C₁-C₆ alkoxy, cycloalkyl, heterocyclyl, or cyano, wherein each of alkyl, heteroalkyl, alkoxy, cycloalkyl and heterocyclyl is independently substituted with 0-5 occurrences of R′;

each R′ is independently selected from C₁-C₆ alkyl, C₁-C₆ heteroalkyl, halo, hydroxyl, cycloalkyl or cyano; or two R′ together with the atom(s) to which they are attached form a cycloalkyl or heterocyclyl ring;

m is 0, 1, or 2; and

n is 0, 1, 2, or 3.

In some embodiments, the compound has structural Formula (Ia):

or a pharmaceutically acceptable salt thereof, wherein ring A, X¹, X², R¹, R², R^(3a), R^(3b), R⁴, R⁵, R⁶, R⁷, R^(8a), R^(8b), R⁹, m and n are as defined as for structural Formula (I).

In some embodiments, the compound has structural Formula (Ib):

or a pharmaceutically acceptable salt thereof, wherein ring A, X¹, X², R¹, R², R^(3a), R^(3b), R⁴, R⁵, R⁶, R⁷, R^(8a), R^(8b), R⁹, m and n are as defined as for structural Formula (I).

In some embodiments of any of structural Formula I, (Ia) or (Ib), R¹ is located at the 5-position. In some embodiments, R¹ is C₁-C₄ alkyl optionally substituted with 0-3 occurrences of R^(a). In some embodiments, m is 1 or 2. In some embodiments, m is 1. In some embodiments, m is 1; R¹ is located at the 5-position; and R¹ is C₁-C₄ alkyl optionally substituted with 0-3 occurrences of R^(a). In some embodiments, R¹ is —CH₃.

In some embodiments of any of structural Formula I or (Ia), R² is selected from hydrogen, hydroxyl, halo and O—C₁-C₄ alkyl. In some embodiments, R² is selected from hydrogen, hydroxyl, fluoro, and —OCH₃.

In some embodiments of any of structural Formula I, (Ia) or (Ib), each of R^(3a), R^(3b), R^(8a) and R^(8b) is independently selected from hydrogen and C₁-C₄ alkyl optionally substituted with 0-3 occurrences of R^(a). In some embodiments, each of R^(3a), R^(3b), R^(8a) and R^(8b) is independently selected from hydrogen and —CH₃. In some embodiments, at least one pair of R^(3a) and R^(3b) or R^(8a) and R^(8b) is simultaneously hydrogen. In some embodiments, each of R^(3a), R^(3b), R^(8a) and R^(8b) is hydrogen.

In some embodiments of any of structural Formula I, (Ia) or (Ib), R⁴ is selected from hydrogen, C₁-C₄ alkyl, and O—C₁-C₄ alkyl, wherein each alkyl portion of R⁴ is optionally substituted with 0-3 occurrences of R^(a). In some embodiments, R⁴ is selected from hydrogen, —CH₃ and —OCH₃.

In some embodiments of any of structural Formula I, (Ia) or (Ib), R⁵ is selected from hydrogen and C₁-C₄ alkyl optionally substituted with 0-3 occurrences of R^(a). In some embodiments, R⁵ is selected from hydrogen and —CH₃.

In some embodiments of any of structural Formula I, (Ia) or (Ib), each R⁶ is independently selected from hydrogen, halo, and C₁-C₄ alkyl substituted with 0-3 occurrences of R^(a). In some embodiments of any of structural Formulae I, (Ia) or (Ib), R⁶ is selected from hydrogen and C₁-C₄ alkyl optionally substituted with 0-3 occurrences of R^(a). In some embodiments, each R⁶ is independently selected from hydrogen, chloro and —CH₃. In some embodiments, each R⁶ is independently selected from hydrogen and —CH₃. In some embodiments, no more than one R⁶ is other than hydrogen.

In some embodiments of any of structural Formula I, (Ia) or (Ib), ring A is a 6-membered monocyclic heteroaryl comprising at least one nitrogen ring atom. In some embodiments, ring A is pyridine or pyrazine. In some embodiments, ring A is selected from

In some embodiments of any of structural Formula I, (Ia) or (Ib), R⁷ is heteroaryl optionally substituted with 0-3 occurrences of R^(b). In some embodiments, R⁷ is a 5-membered monocyclic heteroaryl optionally substituted with 0-3 occurrences of R^(b). In some embodiments, R⁷ is a 5-membered monocyclic nitrogen-containing heteroaryl (e.g., a 5-membered monocyclic heteroaryl comprising 1 or 2 nitrogen atoms) optionally substituted with 0-3 occurrences of R^(b). In some embodiments, R⁷ is pyrazol-1-yl optionally substituted with 0-3 occurrences of R^(b). In some embodiments, n is 1. In some embodiments, n is 1; and R⁷ is pyrazol-1-yl optionally substituted with 0-3 occurrences of R^(b). In some embodiments, R⁷ is 4-fluoropyrazol-1-yl.

In some embodiments of any of structural Formula I, (Ia) or (Ib), R⁹ is selected from hydrogen and C₁-C₄ alkyl optionally substituted with 0-3 occurrences of R^(a). In some embodiments, R⁹ is hydrogen. In some embodiments, R⁹ is C₁-C₄ alkyl. In some embodiments, R⁵ and R⁹ are both hydrogen.

In another aspect, the present invention features a compound having the structural structural Formula (II):

or a pharmaceutically acceptable salt thereof, wherein:

X¹ is selected from N, CH and C(halo);

X² is selected from N and CH;

X³ is selected from N and CH;

R¹² is selected from hydrogen, hydroxyl, halo and O—C₁-C₄ alkyl;

R¹⁴ is selected from hydrogen, —C₁-C₄ alkyl and —O—C₁-C₄ alkyl;

R¹⁵ is selected from hydrogen and —C₁-C₄ alkyl;

R¹⁶ is selected from hydrogen and —C₁-C₄ alkyl;

R¹⁷ is selected from hydrogen and halo; and

R¹⁹ is selected from hydrogen and —C₁-C₄ alkyl.

In some embodiments, —C₁-C₄ alkyl is optionally substituted. In some embodiments, —C₁-C₄ alkyl is substituted. In some embodiments, —C₁-C₄ alkyl is unsubstituted.

In an embodiment, X¹ is N and R¹⁶ is —CH₃.

In some embodiments, the compound is a compound having the structural Formula (IIa):

or a pharmaceutically acceptable salt thereof, wherein X¹, X², X³, R¹², R¹⁴, R¹⁵, R¹⁷, R¹⁶ and R¹⁹ are as defined as for structural Formula (II).

In some embodiments, the compound is a compound having the structural Formula (IIb):

or a pharmaceutically acceptable salt thereof, wherein X¹, X², X³, R¹², R¹⁴, R¹⁵, R¹⁷, R¹⁶ and R¹⁹ are as defined as for structural Formula (II).

In some embodiments of any of structural Formula II, (IIa) or (IIb), X¹ is N or CH. In some embodiments, X¹ is N.

In some embodiments of any of structural Formula II, (IIa) or (IIb), X² is N or CH.

In some embodiments of any of structural Formula II, (IIa) or (IIb), X³ is N. In some embodiments of any of Formula II, (IIa) or (IIb), X³ is CH.

In some embodiments of any of structural Formula II, (IIa) or (IIb), R¹² is selected from hydrogen, hydroxyl, fluoro and —OCH₃. In some embodiments, R¹² is hydrogen. In some embodiments, R¹² is hydroxyl.

In some embodiments of any of structural Formula II, (IIa) or (IIb), R¹⁴ is selected from hydrogen, —CH₃ and —OCH₃. In some embodiments, R¹⁴ is hydrogen. In some embodiments, R¹⁴ is —CH₃.

In some embodiments of any of structural Formula II, (IIa) or (IIb), R¹² is selected from hydrogen and hydroxyl; and R¹⁴ is selected from hydrogen, —CH₃ and —OCH₃. In some embodiments, R¹² and R¹⁴ are both hydrogen.

In some embodiments of any of structural Formula II, (IIa) or (IIb), R¹⁵ is H or —CH₃.

In some embodiments of any of structural Formula II, (IIa) or (IIb), R¹⁶ is selected from hydrogen and —CH₃. In some embodiments, when X¹ is CH, R¹⁶ is —CH₃.

In some embodiments of any of structural Formula II, (IIa) or (IIb), X¹ is N and R¹⁶ is —CH₃.

In some embodiments of any of structural Formula II, (IIa) or (IIb), R¹⁷ is selected from hydrogen and fluoro. In some embodiments, R¹⁷ is hydrogen. In some embodiments, R¹⁷ is fluoro.

In some embodiments of any of structural Formula II, (IIa) or (IIb), R¹⁹ is H or —CH₃. In some embodiments, R¹⁹ is H.

In some embodiments, the present invention features a compound selected from any compound in Table 1.

In another aspect, the present invention features a pharmaceutical composition comprising a compound of structural Formula I, (Ia), (Ib), II, (IIa), or (IIb) described herein (e.g., a compound in Table 1) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In another aspect, the present invention features a method for inhibiting RET activity in a cell or in a patient comprising the step of contacting the cell or administering to the patient a compound described herein (e.g., a compound in Table 1) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

In another aspect, the present invention features a method for treating a subject suffering from a condition mediated by aberrant RET activity, comprising administering to the subject a therapeutically effective amount of a compound described herein (e.g., a compound in Table 1) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

In another aspect, the present invention features a method for treating a subject who has developed resistance to a cancer treatment, comprising administering to the subject a therapeutically effective amount of a compound described herein (e.g., a compound in Table 1) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the subject has developed resistance to a wild-type RET inhibitor.

TABLE 1 Exemplary Compounds of the Invention. Compound Structure 100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

Pharmaceutically acceptable salts of these compounds are also contemplated for the uses described herein.

“Pharmaceutically acceptable salt” refers to any salt of a compound of the invention which retains its biological properties and which is not toxic or otherwise undesirable for pharmaceutical use. Pharmaceutically acceptable salts may be derived from a variety of organic and inorganic counter-ions well known in the art and include. Such salts include: (1) acid addition salts formed with organic or inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic, acetic, trifluoroacetic, trichloroacetic, propionic, hexanoic, cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic, succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric, benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic, phthalic, lauric, methanesulfonic, ethanesulfonic, 1,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic, 4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic, camphoric, camphorsulfonic, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucoheptonic, 3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric, gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic, cyclohexylsulfamic, quinic, muconic acid and the like acids; or (2) salts formed when an acidic proton present in the parent compound either (a) is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion or an aluminum ion, or alkali metal or alkaline earth metal hydroxides, such as sodium, potassium, calcium, magnesium, aluminum, lithium, zinc, and barium hydroxide, ammonia or (b) coordinates with an organic base, such as aliphatic, alicyclic, or aromatic organic amines, such as ammonia, methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N′-dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, N-methylglucamine piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, and the like. Pharmaceutically acceptable salts further include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium and the like, and when the compound contains a basic functionality, salts of non-toxic organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, besylate, acetate, maleate, oxalate and the like.

Pharmaceutical Compositions

Pharmaceutical compositions of the invention comprise one or more compounds of the invention and one or more physiologically or pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof. Each carrier must be “acceptable” in the sense of being compatible with the subject composition and its components and not injurious to the patient. Some examples of materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

The compositions of the invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. In some embodiments, the compositions of the invention are administered orally, intraperitoneally or intravenously. Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tween, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

The pharmaceutically acceptable compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

Alternatively, the pharmaceutically acceptable compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutically acceptable compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutically acceptable compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutically acceptable compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

The pharmaceutically acceptable compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

The amount of the compounds of the present invention that may be combined with the carrier materials to produce a composition in a single dosage form will vary depending upon the host treated, the particular mode of administration.

Dosages

Toxicity and therapeutic efficacy of compounds of the invention, including pharmaceutically acceptable salts and deuterated variants, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The LD₅₀ is the dose lethal to 50% of the population. The ED₅₀ is the dose therapeutically effective in 50% of the population. The dose ratio between toxic and therapeutic effects (LD₅₀/ED₅₀) is the therapeutic index. Compounds that exhibit large therapeutic indexes are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

Data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds may lie within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of a compound of the present invention in the composition will also depend upon the particular compound in the composition.

Treatment

RET fusions have been implicated in several types of cancers. Generally, these RET fusions have a RET kinase domain that is the same as in wild-type RET; therefore, as used herein, any RET protein with the same kinase domain as wild-type RET will be referred to as “wild-type RET” unless noted otherwise. Mutations can occur at least in the RET extracellular and kinase domains. Mutations can occur in the RET kinase domain, leading to resistant mutants of RET.

The activity of exemplary compounds that are approved or in development for RET-related conditions is shown below. As shown, the compounds are active against the wild-type RET, but are much less active against the mutated forms (“wild-type RET inhibitors”).

TABLE 2 RET wt RET V804L Biochemical Biochemical Compound IC₅₀ (nM) IC₅₀ (nM) Cabozantinib 46 219 Vandetanib 1.2 902 Sorafenib 7.9 95.2 Regorafenib 5.1 29.8

The invention provides compounds that inhibit both wild-type RET and mutants of RET, e.g., mutants of RET that are resistant to current standard of care treatments (“RET resistant mutants”). In addition, the compounds of the invention can be selective for wild-type RET, over other kinases, thus leading to reduced toxicities associated with inhibiting other kinases. In one aspect, compounds of the invention are selective for RET over KDR. In one aspect, compounds of the invention do not cause adverse effects such as hypertension, arterial thrombosis, and hemorrhage.

Mutations can be predicted using structural biology and computational analyses, as well as by examining codon sequences in which a sequence change gives rise to a codon for a different amino acid. Using such methods, RET resistant mutants are predicted to have point mutations at the 804 gatekeeper residue in the RET protein and/or at residues at or near the gatekeeper residue. In some embodiments, the mutation may be at one or more of the 804, 806, 810, 865, 870, 891, and 918 residues. Specific examples of RET resistant mutants include: V804L, V804M, V804E, Y806C, Y806S, Y806H, Y806N, G810R, G810S, L865V, L870F, S891A and M918T mutants.

Mutations occurring from administration of a particular inhibitor (e.g., a known RET wild-type inhibitor) can be determined experimentally by exposing cells to a mutation-promoting agent, such as ENU. The cells are washed, then plated with increasing concentrations (2-100× proliferation IC₅₀) of the compound of choice. The wells with cellular outgrowth are then collected after 3-4 weeks. The RET kinase domain is then sequenced to identify resistance mutations (i.e., altered forms of the RET protein that retain enzymatic activity). Resistance can be confirmed by exposing these cells with the compound of choice. Resistant mutants that have been identified experimentally include the V804L, V804E, V804M, and Y806H mutants. In some embodiments, the mutation is a substitution of cysteine (C609, C611, C618, C620, C630, and C634) in the RET extracellular domain for any other amino acid. In some embodiments, the RET cysteine variants (affecting C609, C611, C618, and C620) are the “Janus mutations.” In some embodiments, RET mutations include: RET C634W, RET M918T, V804L, V804E, V804M, V804L, Y806C, Y806S, Y806N, Y806H, G810R, G810S, L865V, L870F, and S891A mutants.

Because of their activity against wild-type RET and mutant RET, the compounds described herein can be used to treat a patient with a condition associated with aberrant RET activity. The compounds described herein can provide treatments for irritable bowel syndrome (IBS), proliferative diseases, and any other conditions related to aberant RET activity. The compounds can be used to treat irritable bowel syndrome. The compounds can be used to treat various cancers. In some embodiments, the cancer is selected from papillary thyroid carcinoma (PTC), medullary thyroid cancer (MTC), pheochromocytoma (PC), pancreatic ductal adenocarcinoma, multiple endocrine neoplasia (MEN2A and MEN2B), metastatic breast cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, chronic myelomonocytic leukemia, colorectal cancer, ovarian cancer, and cancers of the salivary gland. In some embodiments, the cancer is a solid tumor. In some embodiments, the condition associated with aberrant RET activity is a thyroid cancer (e.g., papillary thyroid carcinoma, thyroid adenocarcinoma, or MTC, e.g., familial MTC), lung cancer (e.g., lung adenocarcinoma, small-cell lung carcinoma, or non-small cell lung carcinoma), breast cancer (e.g., estrogen receptor-positive tumors and endocrine-resistant tumors e.g., resistant to oestrogen modulators such as tamoxifen, agents that block oestrogen biosynthesis such as aromatase inhibitors, and oestrogen receptor antagonists such as fulvestrant), pancreatic cancer (e.g., carcinoma of the pancreas or pancreatic ductal carcinoma), haematopoietic cancer, e.g., a leukemia (e.g., chronic myelomonocytic leukemia or acute myeloid leukemia), colon cancer (e.g., colon carcinoma), melanoma (e.g., cutaneous or desmoplastic malignant melanomas), prostate cancer, renal cancer (e.g., renal cell carcinoma), and head and neck tumors, neuroblastoma, ganglioneuroma (e.g., ganglioneuroma of the mouth or gut), colon cancer (e.g., sporadic colon cancers), MEN2A (multiple endocrine neoplasia type 2A), or MEN2B (multiple endocrine neoplasia type 2B). In one aspect, the MEN2A is characterized by MTC and includes adrenal tumor pheochromocytoma. Substitutions of cysteines in RET are found in subjects with MEN2A and also frequent in FMTC. RET extracellular domain exon 8 mutations, such as G533C) or the RET intracellular domain (residues E768, L790, Y791, V804, and S891) are associated with FMTC or MEN2A. Substitutions in the RET kinase domain, Met918 to Thr (M918T) or A883F are found in subjects with MEN2B. RET M918T and RET A883F are also found in sporadic MTC.

The compounds can also be used to treat a patient who has developed resistance to a wild-type RET inhibitor, or a patient with a particular RET mutant. The method includes the step of administering a compound or composition of the invention that is active against one or more RET resistant mutants. In certain embodiments, the RET resistant mutant is selected from V804L, V804M, V804E, Y806C, Y806S, Y806N, Y806H, G810R, G810S, L865V, L870F, S891A and M918T. By “active” is meant that a compound has an IC₅₀ of less than 1 μM, 500 nM, 250 nM, 100 nM, 75 nM, 50 nM, 25 nM, 10 nM, or 5 nM when measured in a biochemical assay, against at least one resistant mutant.

The compounds may also be used to treat a subject having a RET-altered cell, cancer, gene, or gene product. The RET alteration may be, e.g., a point mutation, insertion, deletion, amplification, or fusion, or a combination thereof.

The compounds may also be used to treat a subject having a RET-altered cell, cancer, gene, or gene product comprising a RET alteration described in Table 3 or Table 4 herein. In some embodiments, the subject has a fusion between RET and a RET fusion partner listed in Table 3, e.g., comprises a fusion protein that comprises RET or a fragment thereof and a protein of Table 3 or fragment thereof. In some embodiments, the fusion partner is N-terminal or C-terminal of RET. In some embodiments, the subject has an alteration at a position in RET that is described in Table 4. In some embodiments, a subset of the subject's cells, e.g., a subset of the subject's tumor cells, comprise the RET alteration. In some embodiments, a subset of the subject's cells, e.g., a subset of the subject's tumor cells, are RET-altered. In some embodiments, the subject has a cancer listed in Table 3, e.g., the subject has both a RET mutation and a cancer listed in Table 3.

TABLE 3 RET fusions RET fusion partner Exemplary cancers in which the fusion is found BCR Chronic Myelomonocytic Leukemia (CMML) CLIP 1 Adenocarcinoma KIFSB NSCLC, Ovarian Cancer, Spitzoid Neoplasm; Lung Adenocarcinoma, Adenosquamous Carcinomas CCDC6 NSCLC, Colon Cancer, Papillary Thyroid Cancer; Adenocarcinoma; Lung Adenocarcinoma; Metastatic Colorectal Cancer; Adenosquamous Carcinoma, Metastatic papillary thyroid cancer PTClex9 Metastatic papillary thyroid cancer NCOA4 Papillary Thyroid Cancer, NSCLC, Colon Cancer, Salivary Gland Cancer, Metastatic Colorectal Cancer; Lung Adenocarcinoma, Adenosquamous Carcinomas; Diffuse Sclerosing Variant of Papillary Thyroid Cancer TRIM33 NSCLC, Papillary Thyroid Cancer ERC1 Papillary Thyroid Cancer, Breast Cancer FGFRIOP CMML, Primary Myelofibrosis with secondary Acute Myeloid Leukemia MBD1 Papillary Thyroid Cancer RAB61P2 Papillary Thyroid Cancer PRKAR1A Papillary Thyroid Cancer TRIM24 Papillary Thyroid Cancer KTN1 Papillary Thyroid Cancer GOLGA5 Papillary Thyroid Cancer, Spitzoid Neoplasms HOOK3 Papillary Thyroid Cancer KIAA1468 Papillary Thyroid Cancer, Lung Adenocarcinoma TRIM27 Papillary Thyroid Cancer AKAP13 Papillary Thyroid Cancer FKBP15 Papillary Thyroid Cancer SPECC1L Papillary Thyroid Cancer, Thyroid Gland Carcinoma TBL1XR1 Papillary Thyroid Cancer, Thyroid Gland Carcinoma CEP55 Diffuse Gastric Cancer CUX1 Lung Adenocarcinoma ACBD5 Papillary Thyroid Carcinoma MYH13 Medullary Thyroid Carcinoma PIBF1 Bronchiolus Lung Cell Carcinoma KIAA1217 Papillary Thyroid Cancer, Lung Adenocarcinoma, NSCLC MPRIP NSCLC

TABLE 4 RET mutations Amino acid position 2 Amino acid position 665 (e.g., H665Q) Amino acid position 3 Amino acid position 666 (e.g., K666E, K666M, or K666N) Amino acid position 4 Amino acid position 686 (e.g., S686N) Amino acid position 5 Amino acid position 691 (e.g., G691S) Amino acid position 6 Amino acid position 694 (e.g., R694Q) Amino acid position 7 Amino acid position 700 (e.g., M700L) Amino acid position 8 Amino acid position 706 (e.g., V706M or V706A) Amino acid position 11 Amino acid position 713 splice variant (e.g., E713K) Amino acid position 12 Amino acid position 736 (e.g., G736R) Amino acid position 13 Amino acid position 748 (e.g., G748C) Amino acid position 20 Amino acid position 750 (e.g., A750P) Amino acid position 32 (e.g., S32L) Amino acid position 765 (e.g., S765P) Amino acid position 34 (e.g., D34S) Amino acid position 766 (e.g., P766S or P766M6) Amino acid position 40 (e.g., L40P) Amino acid position 768 (e.g., E768Q or E768D) Amino acid position 64 (e.g., P64L) Amino acid position 769 (e.g., L769L) Amino acid position 67 (e.g., R67H) Amino acid position 770 (e.g., R770Q) Amino acid position 114 (e.g., R114H) Amino acid position 771 (e.g., D771N) Amino acid position 136 (e.g., glutamic Amino acid position 777 (e.g., N777S) acid to stop codon) Amino acid position 145 (e.g., V145G) Amino acid position 778 (e.g., V778I) Amino acid position 180 (e.g., arginine to Amino acid position 781 (e.g., Q781R) stop codon) Amino acid position 200 Amino acid position 790 (e.g., L790F) Amino acid position 292 (e.g., V292M) Amino acid position 791 (e.g., Y791F or Y791N) Amino acid position 294 Amino acid position 802 Amino acid position 321 (e.g., G321R) Amino acid position 804 (e.g., V804L, V804M, V804M*,orV804E) Amino acid position 330 (e.g., R330Q) Amino acid position 805 (e.g., E805K) Amino acid position 338 (e.g., T338I) Amino acid position 806 (e.g., E806C, Y806E, Y806F, Y806S, Y806G, or Y806C) Amino acid position 360 (e.g., R360W) Amino acid position 818 (e.g., E818K) Amino acid position 373 (e.g., alanine to Amino acid position 819 (e.g., S819I) frameshift) Amino acid position 393 (e.g., F393L) Amino acid position 823 (e.g., G823E) Amino acid position 432 Amino acid position 826 (e.g., Y826M) Δ Amino acid residues 505-506 (6-Base Amino acid position 833 (e.g., R833C) Pair In-Frame Germline Deletion in Exon 7) Amino acid position 510 (e.g., A510V) Amino acid position 841 (e.g., P841L or P841P) Amino acid position 511 (e.g., E51IK) Amino acid position 843 (e.g., E843D) Amino acid position 513 (e.g., A513D) Amino acid position 844 (e.g., R844W, R844Q, or R844L) Amino acid position 515 (e.g., C515S, Amino acid position 848 (e.g., M848T) C515W) Amino acid position 525 (e.g., R525W) Amino acid position 852 (e.g., 1852M) Amino acid position 531 (e.g., C531R, or Amino acid position 866 (e.g., A866W) 9 base pair duplication) Amino acid position 532 (e.g., Amino acid position 873 (e.g., R873W) duplication) Amino acid position 533 (e.g., G533C or G533S) Amino acid position 876 (e.g., A876V) Amino acid position 550 (e.g., G550E) Amino acid position 881 (e.g., L881V) Amino acid position 591 (e.g., V591I) Amino acid position 882 Amino acid position 593 (e.g., G593E) Amino acid position 883 (e.g., A883F, A883S, A883T, or A883T*) Amino acid position 600 (e.g., R600Q) Amino acid position 884 (e.g., E884K) Amino acid position 602 (e.g., I602V) Amino acid position 886 (e.g., R886W) Amino acid position 603 (e.g., K603Q or Amino acid position 891 (e.g., S891A) K603E2) Amino acid position 606 (e.g., Y606C) Amino acid position 897 (e.g., R897Q) Amino acid position 609 (e.g., C609Y, Amino acid position 898 (e.g., D898V) C609S, C609G, C609R, C609F, or C609W) Amino acid position 611 (e.g., C611R, Amino acid position 901 (e.g., E901K) C611S, C611G, C611Y, C611F, or C611W) Amino acid position 618 (e.g., C618S, Amino acid position 904 (e.g., S904F or S904C2) C618Y, C618R, C618Y, C618G, C618F, C618W) Amino acid position 619 (e.g., F619F) Amino acid position 907 (e.g., K907E or K907M) Amino acid position 620 (e.g., C620S, Amino acid position 908 (e.g., R908K) C620W, C620R, C620G, C620L, C620Y, C620F) Amino acid position 623 (e.g., E623K) Amino acid position 911 (e.g., G911D) Amino acid position 624 (e.g., D624N) Amino acid position 912 (e.g., R912P, R912Q) Amino acid position 630 (e.g., C630A, Amino acid position 918 (e.g., M918T, M918V, or C630R, C630S, C630Y, or C630F) M918L6) Amino acid position 631 (e.g., D631N, Amino acid position 919 (e.g., A919V) D631Y, D631A, D631G, D631V, or D631E) Amino acid position 632 (e.g., E632K or Amino acid position 921 (e.g., E921K) E632G5) Δ Amino acid residues 632-633 (6-Base Amino acid position 922 (e.g., S922P or S922Y) Pair In-Frame Germline Deletion in Exon 11) Amino acid position 633 (e.g., 9 base pair Amino acid position 930 (e.g., T930M) duplication) Amino acid position 634 (e.g., C634W, Amino acid position 961 (e.g., F961L) C634Y, C634S, C634R, C634F, C634G, C634L, C634A, or C634T, or an insertion ELCR2, or a 12 base pair duplication) Amino acid position 635 (e.g., R635G) Amino acid position 972 (e.g., R972G) Amino acid position 636 (e.g., T636P or Amino acid position 982 (e.g., R982C) T636M4) Amino acid position 640 (e.g., A640G) Amino acid position 1009 (e.g., M 1009V) Amino acid position 641 (e.g., A641S or Amino acid position 1017 (e.g., D1017N) A641T8) Amino acid position 648 (e.g., V6481) Amino acid position 1041 (e.g., V1041G) Amino acid position 649 (e.g., S649L) Amino acid position 1064 (e.g., M1064T) Amino acid position 664 (e.g., A664D) RET + 3

RET has two primary protein and mRNA isoforms, named RET51 and RETS. In embodiments, RET has a sequence of isoform RET51 as shown below as SEQ ID NO: 1. The kinase domain, corresponding to amino acids 724-1016 of SEQ ID NO: 1, is highlighted. Unless otherwise indicated, the amino acid positions described herein refer to the numbering of RET51 shown below.

SEQ ID NO: 1:         10         20         30         40         50 MAKATSGAAG LRLLLLLLLP LLGKVALGLY FSRDAYWEKL YVDQAAGTPL         60         70         80         90        100 LYVHALRDAP EEVPSFRLGQ HLYGTYRTRL HENNWICIQE DTGLLYLNRS        110        120        130        140        150 LDHSSWEKLS VRNRGFPLLT VYLKVFLSPT SLREGECQWP GCARVYFSFF        160        170        180        190        200 NTSFPACSSL KPRELCFPET RPSFRIRENR PPGTFHQFRL LPVQFLCPNI        210        220        230        240        250 SVAYRLLEGE GLPFRCAPDS LEVSTRWALD REQREKYELV AVCTVHAGAR        260        270        280        290        300 EEVVMVPFPV TVYDEDDSAP TFPAGVDTAS AVVEFKRKED TVVATLRVFD        310        320        330        340        350 ADVVPASGEL VRRYTSTLLP GDTWAQQTFR VEHWPNETSV QANGSFVRAT        360        370        380        390        400 VHDYRLVLNR NLSISENRTM QLAVLVNDSD FQGPGAGVLL LHFNVSVLPV        410        420        430        440        450 SLHLPSTYSL SVSRRARRFA QIGKVCVENC QAFSGINVQY KLHSSGANCS        460        470        480        490        500 TLGVVTSAED TSGILFVNDT KALRRPKCAE LHYMVVATDQ QTSRQAQAQL        510        520        530        540        550 LVTVEGSYVA EEAGCPLSCA VSKRRLECEE CGGLGSPTGR CEWRQGDGKG        560        570        580        590        600 ITRNFSTCSP STKTCPDGHC DVVETQDINI CPQDCLRGSI VGGHEPGEPR        610        620        630        640        650 GIKAGYGTCN CFPEEEKCFC EPEDIQDPLC DELCRTVIAA AVLFSFIVSV        660        670        680        690        700 LLSAFCIHCY HKFAHKPPIS SAEMTFRRPA QAFPVSYSSS GARRPSLDSM        710        720        730        740        750 ENQVSVDAFK ILEDPKWEFP RKNLVLGKTL GEGEFGKVVK ATAFHLKGRA        760        770        780        790        800 GYTTVAVKML KENASPSELR DLLSEFNVLK QVNHPHVIKL YGACSQDGPL        810        820        830        840        850 LLIVEYAKYG SLRGFLRESR KVGPGYLGSG GSRNSSSLDH PDERALTMGD        860        870        880        890        900 LISFAWQISQ GMQYLAEMKL VHRDLAARNI LVAEGRKMKI SDFGLSRDVY        910        920        930        940        950 EEDSYVKRSQ GRIPVKWMAI ESLFDHIYTT QSDVWSFGVL LWEIVTLGGN        960        970        980        990       1000 PYPGIPPERL FNLLKTGHRM ERPDNCSEEM YRLMLQCWKQ EPDKRPVFAD       1010       1020       1030       1040       1050 ISKDLEKMMV KRRDYLDLAA STPSDSLIYD DGLSEEETPL VDCNNAPLPR       1060       1070       1080       1090       1100 ALPSTWIENK LYGMSDPNWP GESPVPLTRA DGTNTGFPRY PNDSVYANWM       1110 LSPSAAKLMD TFDS

In embodiments, RET has a sequence of isoform RETS as shown below in SEQ ID NO: 2 (wherein the kinase domain is highlighted)

SEQ ID NO: 2:         10         20         30         40         50 MAKATSGAAG LRLLLLLLLP LLGKVALGLY FSRDAYWEKL YVDQAAGTPL         60         70         80         90        100 LYVHALRDAP EEVPSFRLGQ HLYGTYRTRL HENNWICIQE DTGLLYLNRS        110        120        130        140        150 LDHSSWEKLS VRNRGFPLLT VYLKVFLSPT SLREGECQWP GCARVYFSFF         160        170       180        190        200 NTSFPACSSL KPRELCFPET RPSFRIRENR PPGTFHQFRL LPVQFLCPNI        210        220        230        240        250 SVAYRLLEGE GLPFRCAPDS LEVSTRWALD REQREKYELV AVCTVHAGAR        260        270        280        290        300 EEVVMVPFPV TVYDEDDSAP TFPAGVDTAS AVVEFKRKED TVVATLRVFD        310        320        330        340        350 ADVVPASGEL VRRYTSTLLP GDTWAQQTFR VEHWPNETSV QANGSFVRAT        360        370        380        390        400 VHDYRLVLNR NLSISENRTM QLAVLVNDSD FQGPGAGVLL LHFNVSVLPV        410        420        430        440        450 SLHLPSTYSL SVSRRARRFA QIGKVCVENC QAFSGINVQY KLHSSGANCS        460        470        480        490        500 TLGVVTSAED TSGILFVNDT KALRRPKCAE LHYMVVATDQ QTSRQAQAQL        510        520        530        540        550 LVTVEGSYVA EEAGCPLSCA VSKRRLECEE CGGLGSPTGR CEWRQGDGKG        560        570        580        590        600 ITRNFSTCSP STKTCPDGHC DVVETQDINI CPQDCLRGSI VGGHEPGEPR        610        620        630        640        650 GIKAGYGTCN CFPEEEKCFC EPEDIQDPLC DELCRTVIAA AVLFSFIVSV        660        670        680        690        700 LLSAFCIHCY HKFAHKPPIS SAEMTFRRPA QAFPVSYSSS GARRPSLDSM         710       720        730        740        750 ENQVSVDAFK ILEDPKWEFP RKNLVLGKTL GEGEFGKVVK ATAFHLKGRA        760        770        780        790        800 GYTTVAVKML KENASPSELR DLLSEFNVLK QVNHPHVIKL YGACSQDGPL        810        820        830        840        850 LLIVEYAKYG SLRGFLRESR KVGPGYLGSG GSRNSSSLDH PDERALTMGD        860        870        880        890        900 LISFAWQISQ GMQYLAEMKL VHRDLAARNI LVAEGRKMKI SDFGLSRDVY        910        920        930        940        950 EEDSYVKRSQ GRIPVKWMAI ESLFDHIYTT QSDVWSFGVL LWEIVTLGGN        960        970        980        990       1000 PYPGIPPERL FNLLKTGHRM ERPDNCSEEM YRLMLQCWKQ EPDKRPVFAD       1010       1020       1030       1040       1050 ISKDLEKMMV KRRDYLDLAA STPSDSLIYD DGLSEEETPL VDCNNAPLPR       1060       1070 ALPSTWIENK LYGRISHAFT RF

In embodiments, RET51 is encoded by a nucleic acid having the sequence of

SEQ ID NO: 3:    1 agtcccgcga ccgaagcagg gcgcgcagca gcgctgagtg ccccggaacg tgcgtcgcgc   61 ccccagtgtc cgtcgcgtcc gccgcgcccc gggcggggat ggggcggcca gactgagcgc  121 cgcacccgcc atccagaccc gccggcccta gccgcagtcc ctccagccgt ggccccagcg  181 cgcacgggcg atggcgaagg cgacgtccgg tgccgcgggg ctgcgtctgc tgttgctgct  241 gctgctgccg ctgctaggca aagtggcatt gggcctctac ttctcgaggg atgcttactg  301 ggagaagctg tatgtggacc aggcggccgg cacgcccttg ctgtacgtcc atgccctgcg  361 ggacgcccct gaggaggtgc ccagcttccg cctgggccag catctctacg gcacgtaccg  421 cacacggctg catgagaaca actggatctg catccaggag gacaccggcc tcctctacct  481 taaccggagc ctggaccata gctcctggga gaagctcagt gtccgcaacc gcggctttcc  541 cctgctcacc gtctacctca aggtcttcct gtcacccaca tcccttcgtg agggcgagtg  601 ccagtggcca ggctgtgccc gcgtatactt ctccttcttc aacacctcct ttccagcctg  661 cagctccctc aagccccggg agctctgctt cccagagaca aggccctcct tccgcattcg  721 ggagaaccga cccccaggca ccttccacca gttccgcctg ctgcctgtgc agttcttgtg  781 ccccaacatc agcgtggcct acaggctcct ggagggtgag ggtctgccct tccgctgcgc  841 cccggacagc ctggaggtga gcacgcgctg ggccctggac cgcgagcagc gggagaagta  901 cgagctggtg gccgtgtgca ccgtgcacgc cggcgcgcgc gaggaggtgg tgatggtgcc  961 cttcccggtg accgtgtacg acgaggacga ctcggcgccc accttccccg cgggcgtcga 1021 caccgccagc gccgtggtgg agttcaagcg gaaggaggac accgtggtgg ccacgctgcg 1081 tgtcttcgat gcagacgtgg tacctgcatc aggggagctg gtgaggcggt acacaagcac 1141 gctgctcccc ggggacacct gggcccagca gaccttccgg gtggaacact ggcccaacga 1201 gacctcggtc caggccaacg gcagcttcgt gcgggcgacc gtacatgact ataggctggt 1261 tctcaaccgg aacctctcca tctcggagaa ccgcaccatg cagctggcgg tgctggtcaa 1321 tgactcagac ttccagggcc caggagcggg cgtcctcttg ctccacttca acgtgtcggt 1381 gctgccggtc agcctgcacc tgcccagtac ctactccctc tccgtgagca ggagggctcg 1441 ccgatttgcc cagatcggga aagtctgtgt ggaaaactgc caggcattca gtggcatcaa 1501 cgtccagtac aagctgcatt cctctggtgc caactgcagc acgctagggg tggtcacctc 1561 agccgaggac acctcgggga tcctgtttgt gaatgacacc aaggccctgc ggcggcccaa 1621 gtgtgccgaa cttcactaca tggtggtggc caccgaccag cagacctcta ggcaggccca 1681 ggcccagctg cttgtaacag tggaggggtc atatgtggcc gaggaggcgg gctgccccct 1741 gtcctgtgca gtcagcaaga gacggctgga gtgtgaggag tgtggcggcc tgggctcccc 1801 aacaggcagg tgtgagtgga ggcaaggaga tggcaaaggg atcaccagga acttctccac 1861 ctgctctccc agcaccaaga cctgccccga cggccactgc gatgttgtgg agacccaaga 1921 catcaacatt tgccctcagg actgcctccg gggcagcatt gttgggggac acgagcctgg 1981 ggagccccgg gggattaaag ctggctatgg cacctgcaac tgcttccctg aggaggagaa 2041 gtgcttctgc gagcccgaag acatccagga tccactgtgc gacgagctgt gccgcacggt 2101 gatcgcagcc gctgtcctct tctccttcat cgtctcggtg ctgctgtctg ccttctgcat 2161 ccactgctac cacaagtttg cccacaagcc acccatctcc tcagctgaga tgaccttccg 2221 gaggcccgcc caggccttcc cggtcagcta ctcctcttcc ggtgcccgcc ggccctcgct 2281 ggactccatg gagaaccagg tctccgtgga tgccttcaag atcctggagg atccaaagtg 2341 ggaattccct cggaagaact tggttcttgg aaaaactcta ggagaaggcg aatttggaaa 2401 agtggtcaag gcaacggcct tccatctgaa aggcagagca gggtacacca cggtggccgt 2461 gaagatgctg aaagagaacg cctccccgag tgagcttcga gacctgctgt cagagttcaa 2521 cgtcctgaag caggtcaacc acccacatgt catcaaattg tatggggcct gcagccagga 2581 tggcccgctc ctcctcatcg tggagtacgc caaatacggc tccctgcggg gcttcctccg 2641 cgagagccgc aaagtggggc ctggctacct gggcagtgga ggcagccgca actccagctc 2701 cctggaccac ccggatgagc gggccctcac catgggcgac ctcatctcat ttgcctggca 2761 gatctcacag gggatgcagt atctggccga gatgaagctc gttcatcggg acttggcagc 2821 cagaaacatc ctggtagctg aggggcggaa gatgaagatt tcggatttcg gcttgtcccg 2881 agatgtttat gaagaggatt cctacgtgaa gaggagccag ggtcggattc cagttaaatg 2941 gatggcaatt gaatcccttt ttgatcatat ctacaccacg caaagtgatg tatggtcttt 3001 tggtgtcctg ctgtgggaga tcgtgaccct agggggaaac ccctatcctg ggattcctcc 3061 tgagcggctc ttcaaccttc tgaagaccgg ccaccggatg gagaggccag acaactgcag 3121 cgaggagatg taccgcctga tgctgcaatg ctggaagcag gagccggaca aaaggccggt 3181 gtttgcggac atcagcaaag acctggagaa gatgatggtt aagaggagag actacttgga 3241 ccttgcggcg tccactccat ctgactccct gatttatgac gacggcctct cagaggagga 3301 gacaccgctg gtggactgta ataatgcccc cctccctcga gccctccctt ccacatggat 3361 tgaaaacaaa ctctatggca tgtcagaccc gaactggcct ggagagagtc ctgtaccact 3421 cacgagagct gatggcacta acactgggtt tccaagatat ccaaatgata gtgtatatgc 3481 taactggatg ctttcaccct cagcggcaaa attaatggac acgtttgata gttaacattt 3541 ctttgtgaaa ggtaatggac tcacaagggg aagaaacatg ctgagaatgg aaagtctacc 3601 ggccctttct ttgtgaacgt cacattggcc gagccgtgtt cagttcccag gtggcagact 3661 cgtttttggt agtttgtttt aacttccaag gtggttttac ttctgatagc cggtgatttt 3721 ccctcctagc agacatgcca caccgggtaa gagctctgag tcttagtggt taagcattcc 3781 tttctcttca gtgcccagca gcacccagtg ttggtctgtg tccatcagtg accaccaaca 3841 ttctgtgttc acatgtgtgg gtccaacact tactacctgg tgtatgaaat tggacctgaa 3901 ctgttggatt tttctagttg ccgccaaaca aggcaaaaaa atttaaacat gaagcacaca 3961 cacaaaaaag gcagtaggaa aaatgctggc cctgatgacc tgtccttatt cagaatgaga 4021 gactgcgggg ggggcctggg ggtagtgtca atgcccctcc agggctggag gggaagaggg 4081 gccccgagga tgggcctggg ctcagcattc gagatcttga gaatgatttt tttttaatca 4141 tgcaaccttt ccttaggaag acatttggtt ttcatcatga ttaagatgat tcctagattt 4201 agcacaatgg agagattcca tgccatcttt actatgtgga tggtggtatc agggaagagg 4261 gctcacaaga cacatttgtc ccccgggccc accacatcat cctcacgtgt tcggtactga 4321 gcagccacta cccctgatga gaacagtatg aagaaagggg gctgttggag tcccagaatt 4381 gctgacagca gaggctttgc tgctgtgaat cccacctgcc accagcctgc agcacacccc 4441 acagccaagt agaggcgaaa gcagtggctc atcctacctg ttaggagcag gtagggcttg 4501 tactcacttt aatttgaatc ttatcaactt actcataaag ggacaggcta gctagctgtg 4561 ttagaagtag caatgacaat gaccaaggac tgctacacct ctgattacaa ttctgatgtg 4621 aaaaagatgg tgtttggctc ttatagagcc tgtgtgaaag gcccatggat cagctcttcc 4681 tgtgtttgta atttaatgct gctacaagat gtttctgttt cttagattct gaccatgact 4741 cataagcttc ttgtcattct tcattgcttg tttgtggtca cagatgcaca acactcctcc 4801 agtcttgtgg gggcagcttt tgggaagtct cagcagctct tctggctgtg ttgtcagcac 4861 tgtaacttcg cagaaaagag tcggattacc aaaacactgc ctgctcttca gacttaaagc 4921 actgatagga cttaaaatag tctcattcaa atactgtatt ttatataggc atttcacaaa 4981 aacagcaaaa ttgtggcatt ttgtgaggcc aaggcttgga tgcgtgtgta atagagcctt 5041 gtggtgtgtg cgcacacacc cagagggaga gtttgaaaaa tgcttattgg acacgtaacc 5101 tggctctaat ttgggctgtt tttcagatac actgtgataa gttcttttac aaatatctat 5161 agacatggta aacttttggt tttcagatat gcttaatgat agtcttacta aatgcagaaa 5221 taagaataaa ctttctcaaa ttattaaaaa tgcctacaca gtaagtgtga attgctgcaa 5281 caggtttgtt ctcaggaggg taagaactcc aggtctaaac agctgaccca gtgatgggga 5341 atttatcctt gaccaattta tccttgacca ataacctaat tgtctattcc tgagttataa 5401 aagtccccat ccttattagc tctactggaa ttttcataca cgtaaatgca gaagttacta 5461 agtattaagt attactgagt attaagtagt aatctgtcag ttattaaaat ttgtaaaatc 5521 tatttatgaa aggtcattaa accagatcat gttccttttt ttgtaatcaa ggtgactaag 5581 aaaatcagtt gtgtaaataa aatcatgtat cataaaaaaa aaaaaaaaa //

In embodiments, RETS is encoded by a nucleic acid having the sequence of

SEQ ID NO: 4:    1 agtcccgcga ccgaagcagg gcgcgcagca gcgctgagtg ccccggaacg tgcgtcgcgc   61 ccccagtgtc cgtcgcgtcc gccgcgcccc gggcggggat ggggcggcca gactgagcgc  121 cgcacccgcc atccagaccc gccggcccta gccgcagtcc ctccagccgt ggccccagcg  181 cgcacgggcg atggcgaagg cgacgtccgg tgccgcgggg ctgcgtctgc tgttgctgct  241 gctgctgccg ctgctaggca aagtggcatt gggcctctac ttctcgaggg atgcttactg  301 ggagaagctg tatgtggacc aggcggccgg cacgcccttg ctgtacgtcc atgccctgcg  361 ggacgcccct gaggaggtgc ccagcttccg cctgggccag catctctacg gcacgtaccg  421 cacacggctg catgagaaca actggatctg catccaggag gacaccggcc tcctctacct  481 taaccggagc ctggaccata gctcctggga gaagctcagt gtccgcaacc gcggctttcc  541 cctgctcacc gtctacctca aggtcttcct gtcacccaca tcccttcgtg agggcgagtg  601 ccagtggcca ggctgtgccc gcgtatactt ctccttcttc aacacctcct ttccagcctg  661 cagctccctc aagccccggg agctctgctt cccagagaca aggccctcct tccgcattcg  721 ggagaaccga cccccaggca ccttccacca gttccgcctg ctgcctgtgc agttcttgtg  781 ccccaacatc agcgtggcct acaggctcct ggagggtgag ggtctgccct tccgctgcgc  841 cccggacagc ctggaggtga gcacgcgctg ggccctggac cgcgagcagc gggagaagta  901 cgagctggtg gccgtgtgca ccgtgcacgc cggcgcgcgc gaggaggtgg tgatggtgcc  961 cttcccggtg accgtgtacg acgaggacga ctcggcgccc accttccccg cgggcgtcga 1021 caccgccagc gccgtggtgg agttcaagcg gaaggaggac accgtggtgg ccacgctgcg 1081 tgtcttcgat gcagacgtgg tacctgcatc aggggagctg gtgaggcggt acacaagcac 1141 gctgctcccc ggggacacct gggcccagca gaccttccgg gtggaacact ggcccaacga 1201 gacctcggtc caggccaacg gcagcttcgt gcgggcgacc gtacatgact ataggctggt 1261 tctcaaccgg aacctctcca tctcggagaa ccgcaccatg cagctggcgg tgctggtcaa 1321 tgactcagac ttccagggcc caggagcggg cgtcctcttg ctccacttca acgtgtcggt 1381 gctgccggtc agcctgcacc tgcccagtac ctactccctc tccgtgagca ggagggctcg 1441 ccgatttgcc cagatcggga aagtctgtgt ggaaaactgc caggcattca gtggcatcaa 1501 cgtccagtac aagctgcatt cctctggtgc caactgcagc acgctagggg tggtcacctc 1561 agccgaggac acctcgggga tcctgtttgt gaatgacacc aaggccctgc ggcggcccaa 1621 gtgtgccgaa cttcactaca tggtggtggc caccgaccag cagacctcta ggcaggccca 1681 ggcccagctg cttgtaacag tggaggggtc atatgtggcc gaggaggcgg gctgccccct 1741 gtcctgtgca gtcagcaaga gacggctgga gtgtgaggag tgtggcggcc tgggctcccc 1801 aacaggcagg tgtgagtgga ggcaaggaga tggcaaaggg atcaccagga acttctccac 1861 ctgctctccc agcaccaaga cctgccccga cggccactgc gatgttgtgg agacccaaga 1921 catcaacatt tgccctcagg actgcctccg gggcagcatt gttgggggac acgagcctgg 1981 ggagccccgg gggattaaag ctggctatgg cacctgcaac tgcttccctg aggaggagaa 2041 gtgcttctgc gagcccgaag acatccagga tccactgtgc gacgagctgt gccgcacggt 2101 gatcgcagcc gctgtcctct tctccttcat cgtctcggtg ctgctgtctg ccttctgcat 2161 ccactgctac cacaagtttg cccacaagcc acccatctcc tcagctgaga tgaccttccg 2221 gaggcccgcc caggccttcc cggtcagcta ctcctcttcc ggtgcccgcc ggccctcgct 2281 ggactccatg gagaaccagg tctccgtgga tgccttcaag atcctggagg atccaaagtg 2341 ggaattccct cggaagaact tggttcttgg aaaaactcta ggagaaggcg aatttggaaa 2401 agtggtcaag gcaacggcct tccatctgaa aggcagagca gggtacacca cggtggccgt 2461 gaagatgctg aaagagaacg cctccccgag tgagcttcga gacctgctgt cagagttcaa 2521 cgtcctgaag caggtcaacc acccacatgt catcaaattg tatggggcct gcagccagga 2581 tggcccgctc ctcctcatcg tggagtacgc caaatacggc tccctgcggg gcttcctccg 2641 cgagagccgc aaagtggggc ctggctacct gggcagtgga ggcagccgca actccagctc 2701 cctggaccac ccggatgagc gggccctcac catgggcgac ctcatctcat ttgcctggca 2761 gatctcacag gggatgcagt atctggccga gatgaagctc gttcatcggg acttggcagc 2821 cagaaacatc ctggtagctg aggggcggaa gatgaagatt tcggatttcg gcttgtcccg 2881 agatgtttat gaagaggatt cctacgtgaa gaggagccag ggtcggattc cagttaaatg 2941 gatggcaatt gaatcccttt ttgatcatat ctacaccacg caaagtgatg tatggtcttt 3001 tggtgtcctg ctgtgggaga tcgtgaccct agggggaaac ccctatcctg ggattcctcc 3061 tgagcggctc ttcaaccttc tgaagaccgg ccaccggatg gagaggccag acaactgcag 3121 cgaggagatg taccgcctga tgctgcaatg ctggaagcag gagccggaca aaaggccggt 3181 gtttgcggac atcagcaaag acctggagaa gatgatggtt aagaggagag actacttgga 3241 ccttgcggcg tccactccat ctgactccct gatttatgac gacggcctct cagaggagga 3301 gacaccgctg gtggactgta ataatgcccc cctccctcga gccctccctt ccacatggat 3361 tgaaaacaaa ctctatggta gaatttccca tgcatttact agattctagc accgctgtcc 3421 cctctgcact atccttcctc tctgtgatgc tttttaaaaa tgtttctggt ctgaacaaaa 3481 ccaaagtctg ctctgaacct ttttatttgt aaatgtctga ctttgcatcc agtttacatt 3541 taggcattat tgcaactatg tttttctaaa aggaagtgaa aataagtgta attaccacat 3601 tgcccagcaa cttaggatgg tagaggaaaa aacagatcag ggcggaactc tcaggggaga 3661 ccaagaacag gttgaataag gcgcttctgg ggtgggaatc aagtcatagt acttctactt 3721 taactaagtg gataaatata caaatctggg gaggtattca gttgagaaag gagccaccag 3781 caccactcag cctgcactgg gagcacagcc aggttccccc agacccctcc tgggcaggca 3841 ggtgcctctc agaggccacc cggcactggc gagcagccac tggccaagcc tcagccccag 3901 tcccagccac atgtcctcca tcaggggtag cgaggttgca ggagctggct ggccctggga 3961 ggacgcaccc ccactgctgt tttcacatcc tttcccttac ccaccttcag gacggttgtc 4021 acttatgaag tcagtgctaa agctggagca gttgcttttt gaaagaacat ggtctgtggt 4081 gctgtggtct tacaatggac agtaaatatg gttcttgcca aaactccttc ttttgtcttt 4141 gattaaatac tagaaattta aaaaaaaaaa aaaa //

The compounds and compositions described herein can be administered alone or in combination with other compounds, including other RET-modulating compounds, or other therapeutic agents. In some embodiments, the compound or composition of the invention may be administered in combination with one or more compounds selected from Cabozantinib (COMETRIQ), Vandetanib (CALPRESA), Sorafenib (NEXAVAR), Sunitinib (SUTENT), Regorafenib (STAVARGA), Ponatinib (ICLUSIG), Bevacizumab (AVASTIN), Crizotinib (XALKORI), or Gefitinib (IRESSA). The compound or composition of the invention may be administered simultaneously or sequentially with the other therapeutic agent by the same of different routes of administration. The compound of the invention may be included in a single formulation with the other therapeutic agent or in separate formulations.

EXAMPLES

The following examples are intended to be illustrative, and are not meant in any way to be limiting.

Synthesis

Compounds of the invention, including salts and N-oxides thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes, such as those in the Schemes below. The reactions for preparing compounds of the invention can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, e.g., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected by the skilled artisan.

Preparation of compounds of the invention can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th ed., John Wiley & Sons: New Jersey, (2006), which is incorporated herein by reference in its entirety.

Reactions can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance (NMR) spectroscopy (e.g., ¹H or ¹³C), infrared (IR) spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry (MS), or by chromatographic methods such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC). Analytical instruments and methods for compound characterization:

LC-MS: Unless otherwise indicated, all liquid chromatography-mass spectrometry (LC-MS) data (sample analyzed for purity and identity) were obtained with an Agilent model-1260 LC system using an Agilent model 6120 mass spectrometer utilizing ES-API ionization fitted with an Agilent Poroshel 120 (EC-C18, 2.7 um particle size, 3.0×50 mm dimensions) reverse-phase column at 22.4 degrees Celsius. The mobile phase consisted of a mixture of solvent 0.1% formic acid in water and 0.1% formic acid in acetonitrile. A constant gradient from 95% aqueous/5% organic to 5% aqueous/95% organic mobile phase over the course of 4 minutes was utilized. The flow rate was constant at 1 mL/min.

Prep LC-MS: Preparative HPLC was performed on a Shimadzu Discovery VP® Preparative system fitted with a Luna 5u C18(2) 100A, AXIA packed, 250×21.2 mm reverse-phase column at 22.4 degrees Celsius. The mobile phase consisted of a mixture of solvent 0.1% formic acid in water and 0.1% formic acid in acetonitrile. A constant gradient from 95% aqueous/5% organic to 5% aqueous/95% organic mobile phase over the course of 25 minutes was utilized. The flow rate was constant at 20 mL/min. Reactions carried out in a microwave were done so in a Biotage Initiator microwave unit.

Silica gel chromatography: Silica gel chromatography was performed on either a Teledyne Isco CombiFlash® Rf unit or a Biotage® Isolera Four unit.

Proton NMR: Unless otherwise indicated, all ¹H NMR spectra were obtained with a Varian 400 MHz Unity Inova 400 MHz NMR instrument (acquisition time=3.5 seconds with a 1 second delay; 16 to 64 scans). Where characterized, all protons were reported in DMSO-d6 solvent as parts-per million (ppm) with respect to residual DMSO (2.50 ppm).

The below Schemes are meant to provide general guidance in connection with preparing the compounds of the invention. One skilled in the art would understand that the preparations shown in the Schemes can be modified or optimized using general knowledge of organic chemistry to prepare various compounds of the invention.

Synthetic Protocol 1:

An aryl dihalide can be treated with an organolithium or organomagnesium halide reagent, such as n-BuLi or i-PrMgCl, and the arylmetal reagent can then undergo addition to an ester substituted cyclobutanone (either commercially available or prepared as described in “Synthesis of Ketone Intermediates” in Example 6). The remaining halide can then undergo a coupling reaction with an arylamine under SnAr conditions or metal-catalyzed coupling conditions to give a tricyclic ester intermediate. The ester can then be hydrolyzed under basic conditions to give an acid, which can then undergo an amide coupling reaction with an amine (either commercially available or prepared as described in “Synthesis of Amine Intermediates” in Example 8). The amides are examples of RET inhibitors described herein, but can also be further modified. For example, the tertiary alcohol can be treated with a fluorinating reagent such as DAST to give a mixture of deoxyfluorinated products which are also examples of RET inhibitors.

Synthetic Protocol 2:

A substituted cycloalkyl iodide (either commercially available or prepared as described in “Synthesis of Iodide Intermediates”) is treated with activated zinc. The zinc could be activated by a variety methods, including but not limited to the method of Reike or treatment with TMS-Cl and 1,2-dibromoethane. The cycloalkyl zinc reagent can then be coupled to a heteroaryl dihalide with palladium catalysis under Negishi coupling conditions. The resulting carbon-carbon can form to give a mixture of diastereomers, which can be separated by chromatography or other separation techniques at this stage. Alternatively the diastereomers can be used as a mixture in the subsequent transformations and separated at a different stage. The remaining heteroaryl halide can then undergo displacement with an aryl amine under either SnAr conditions or palladium mediated coupling conditions. The tricyclic carboxylic ester can then be hydrolyzed under acidic or basic conditions to provide a carboxylic acid intermediate. The carboxylic acid intermediate can then be coupled to a variety of amines, such as those described below under the heading “Synthesis of Amine Intermediates” in Example 8, to provide the amide final product.

Synthetic Protocol 3:

A heteroaryl dihalide can be coupled to an amino pyrazole under nucleophilic aromatic substitution reaction conditions using a base such as diisopropylethylamine (DIPEA) or triethylamine (TEA) in a polar solvent to provide the bicyclic ring system. The bicyclic heteroaryl halide can then be coupled to an excess of alkyl zinc reagent (typically 2 equivalents)(M=Zn) via a palladium-mediated coupling reaction, e.g. Negishi coupling, to provide the tricyclic ring system. The alkylzinc reagents are prepared from zinc insertion into the carbon-halide bond of an ester-substituted alkyl halide, which are either commercially available or prepared as described in “Synthesis of Iodide Intermediates”. The coupling reaction can give a mixture of diastereoisomers, which in some instances can be separated directly and in other instances are separated at a further stage. The carboxylic ester can then be hydrolyzed under acidic or basic conditions to provide a carboxylic acid intermediate. The carboxylic acid intermediate can then be coupled to a variety of amines, such as those described below under the heading “Synthesis of Amine Intermediates,” in Example 8 to provide the amide final product.

Example 1. General Synthesis of Compound 109 and Related Analogs Step 1: Synthesis of methyl 3-(6-bromo-4-methylpyridin-2-yl)-3-hydroxycyclobutanecarboxylate

A solution of 2,6-dibromo-4-methylpyridine (1.50 g, 5.97 mmol) in DCM (30 mL) was cooled to −78° C., and n-BuLi (2.5 M, 2.60 mL, 6.56 mmol) was added dropwise to the above solution at −78° C. The solution was stirred at −78° C. for another 15 min. Methyl 3-oxocyclobutanecarboxylate (917 mg, 7.16 mmol) was added to the solution, and the resultant mixture was stirred at −78° C. for 30 min. The mixture was then quenched by addition of saturated aqueous NH₄Cl solution and extracted with DCM. Organic layers were combined, dried over sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column (PE:EA=2:1) to give methyl 3-(6-bromo-4-methylpyridin-2-yl)-3-hydroxycyclobutanecarboxylate (1.0 g, 56%) as a white solid, MS (ES+) C₁₂H₁₄BrNO₃ requires: 299, found: 300 [M+H]⁺.

Step 2: Synthesis of 3-hydroxy-3-(4-methyl-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)cyclobutanecarboxylate

A mixture of methyl 3-(6-bromo-4-methylpyridin-2-yl)-3-hydroxycyclobutanecarboxylate (400 mg, 1.33 mmol), 5-methyl-1H-pyrazol-3-amine (258 mg, 2.66 mmol), t-BuXPhos (69 mg, 0.40 mmol), Pd₂(dba)₃ (182 mg, 0.20 mmol) and KOAc (391 mg, 3.99 mmol) in DMA (6 mL) was heated to 140° C. for 1 h under microwave irradiation. After cooling to ambient temperature, the mixture was concentrated and purified by silica gel column (PE:EA=1:2) to give 3-hydroxy-3-(4-methyl-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)cyclobutanecarboxylate (120 mg, 29%) as a white solid. MS (ES+) C₁₆H₂₀N₄O₃ requires: 316, found: 317 [M+H]⁺.

Step 3: Synthesis of 3-hydroxy-3-(4-methyl-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)cyclobutanecarboxylate

To a solution of 3-hydroxy-3-(4-methyl-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)cyclobutanecarboxylate (120 mg, 0.38 mmol) in MeOH (5 mL) was added 2 M aqueous LiOH (0.5 mL, 1 mmol) at 25° C. The solution was stirred at 25° C. for 15 h. The solution was concentrated to remove MeOH. The water solution was acidified by 2 M HCl to bring pH to 6. The precipitated solid was collected and dried to give 3-hydroxy-3-(4-methyl-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)cyclobutanecarboxylate (100 mg, 88%) as a yellow solid. MS (ES+) C₁₅H₁₈N₄O₃ requires: 302, found: 303 [M+H]⁺.

Step 4: Synthesis of (1S,4R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-4-hydroxy-4-(4-methyl-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)cyclohexanecarboxamide (Compound 109)

A mixture of 3-hydroxy-3-(4-methyl-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)cyclobutanecarboxylate (100 mg, 0.33 mmol), (S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethanamine hydrochloride (80 mg, 0.33 mmol), HATU (125 mg, 0.33 mmol) and DIEA (128 mg, 0.99 mmol) in DMA (3 mL) was stirred 25° C. for 2 h. The solution was concentrated and purified by preparative HPLC to give the title product (60 mg, 37%) as a white solid. MS (ES+) C₂₅H₂₇FN₈O₂ requires: 490, found: 491 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆) δ ppm 11.70 (s, 1H), 8.86 (s, 1H), 8.68 (d, 1H, J=4.8 Hz), 8.39 (d, 1H, J=0.8 Hz), 8.35 (d, 1H, J=8.0 Hz), 7.95-7.86 (m, 3H), 6.94 (s, 1H), 6.74 (s, 1H), 6.14 (s, 1H), 5.68 (s, 1H), 5.03-5.00 (m, 1H), 3.00-2.98 (m, 1H), 2.63-2.55 (m, 2H), 2.42-2.32 (m, 2H), 2.21 (s, 3H), 2.18 (s, 3H), 1.39 (d, 3H, J=7.2 Hz).

Example 2. General Synthesis of Compound 113 and Related Analogs Step 1: Synthesis of (1S,3R)-3-fluoro-N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-3-(4-methyl-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)cyclobutanecarboxamide

A mixture of (1S,3R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-3-hydroxy-3-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyridin-2-yl)cyclobutanecarboxamide (40 mg, 0.082 mmol) in DCM (4 mL) was cooled to 0° C. DAST (39 mg, 0.24 mmol) was added to the mixture at 0° C. The resultant mixture was stirred at 25° C. for 1 h. The mixture was concentrated and purified by Prep-HPLC to give the title compound (15.6 mg, 39%) as a white solid. MS (ES+) C₂₅H₂₆F₂N₈O requires: 492, found: 493 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆) δ ppm 12.50-11.50 (br, 1H), 10.00-9.00 (br, 1H), 8.66 (d, 1H, J=4.0 Hz), 8.56 (d, 1H, J=6.0 Hz), 8.39 (d, 1H, J=2.0 Hz), 7.94-7.85 (m, 3H), 6.82 (s, 1H), 6.69 (s, 1H), 6.34 (s, 1H), 5.09-5.05 (m, 1H), 3.46-3.40 (m, 2H), 3.29-2.95 (m, 2H), 2.55-2.50 (m, 1H), 2.24 (s, 3H), 2.16 (s, 3H), 1.42 (d, 3H, J=7.2 Hz).

Example 3. Synthesis of Compound 117 and Related Analogs Step 1: Synthesis of methyl 3-(6-bromo-4-methylpyridin-2-yl)-3-methoxycyclobutane-1-carboxylate

A mixture of methyl 3-(6-bromo-4-methylpyridin-2-yl)-3-hydroxycyclobutanecarboxylate (2.2 g, 7.3 mmol) in DMF (66 mL) was cooled to 0° C. Sodium hydride (60% dispersion in oil, 1.45 g, 36.6 mmol) min was added, and after 15 min iodomethane (2.08 g, 14.7 mmol) was added. The cooling bath was removed and the reaction mixture was stirred for 3 h at ambient temperature. The mixture was then partitioned between ethyl acetate and water, and the organic layer was washed with brine. The washed organic layer was dried over sodium sulfate, filtered, and the filtrate was concentrated. The residue was purified by flash-column chromatography on silica gel (gradient elution, 0 to 100% ethyl acetate-hexanes) to give methyl 3-(6-bromo-4-methylpyridin-2-yl)-3-methoxycyclobutane-1-carboxylate (1.6 g, 70%).

Steps 2-4: Synthesis of (1S,3R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-3-methoxy-3-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyridin-2-yl)cyclobutane-1-carboxamide

The title compound was prepared from methyl 3-(6-bromo-4-methylpyridin-2-yl)-3-methoxycyclobutane-1-carboxylate using the procedures described in Synthetic Protocol 1 and Example 1. MS (ES+) C₂₆H₂₉FN₈O₂ requires: 504, found: 505 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆) δ 8.68 (d, J=4.5 Hz, 1H), 8.51 (d, J=7.7 Hz, 1H), 8.40 (d, J=1.7 Hz, 1H), 7.95-7.88 (m, 3H), 7.25 (s, 1H), 7.07 (s, 1H), 5.98 (s, 1H), 5.03-4.98 (m, 1H), 3.07 (s, 3H), 2.97-2.83 (m, 2H), 2.58 (t, J=9.2 Hz, 3H), 2.49 (s, 3H), 2.27 (s, 3H), 1.41 (d, J=7.0 Hz, 3H).

Example 4. General Synthesis of Compound 122 and Related Analogs Step 1: Synthesis of ethyl (1s,3s)-3-(2-chloro-6-methylpyrimidin-4-yl)-1-methoxycyclobutane-1-carboxylate

Ethyl 3-iodo-1-methoxycyclobutane-1-carboxylate (1.00 g, 3.52 mmol) was dissolved in dimethylacetamide (11.7 mL) in a pressure vessel under a stream of nitrogen. Rieke Zinc (5.1 mL of a 50 mg/mL suspension in THF, 3.87 mmol) was added quickly via syringe. The vessel was capped and stirred at ambient temperature for 15 minutes. The vessel was opened under a stream of nitrogen and 2,4-dichloro-6-methylpyrimidine (689 mg, 4.22 mmol) was added followed by PdCl₂dppf (258 mg, 0.352 mmol). The vessel was capped and heated to 70° C. for 3 h. LCMS of the crude reaction mixture showed a ˜3:1 ratio of product isomers. The reaction mixture was then cooled to room temperature, diluted with ethyl acetate, and filtered through celite. The filtrate was transferred to a separatory funnel and washed with water (3×), brine, and dried over sodium sulfate. The dried solution was filtered, and the filtrate was concentrated. The residue was purified by flash-column chromatography on silica gel (gradient elution, 0 to 50% ethyl acetate-hexanes) to give ethyl (1S,3S)-3-(2-chloro-6-methylpyrimidin-4-yl)-1-methoxycyclobutane-1-carboxylate (323 mg, 32%) as a colorless oil. The minor isomer was discarded. MS (ES+) C₁₅H₂₂N₂O₃S requires: 310, found: 311 [M+H]⁺.

Steps 2-4: Synthesis of (1R,3S)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-3-(6-methyl-2-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-4-yl)cyclobutane-1-carboxamide

The title compound was prepared from ethyl (1S,3S)-3-(2-chloro-6-methylpyrimidin-4-yl)-1-methoxycyclobutane-1-carboxylate (192 mg, 0.53 mmol) using the same three-step procedure (cross-coupling, hydrolysis, and amide coupling) as in Synthetic Protocol 1 and Example 1 to give a white solid. MS (ES+) C₂₅H₂₈FN₉O₂ requires: 505, found: 506 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d6) δ 11.77 (s, 1H), 9.24 (s, 1H), 8.67 (dd, J=4.5, 0.8 Hz, 1H), 8.52 (d, J=8.2 Hz, 1H), 8.44 (d, J=2.2 Hz, 1H), 7.99 (dd, J=8.6, 2.3 Hz, 1H), 7.94-7.74 (m, 2H), 6.54 (s, 2H), 5.17-5.01 (m, 1H), 3.16-3.03 (m, 4H), 2.71-2.60 (m, 1H), 2.57-2.36 (m, 4H), 2.26 (s, 3H), 2.17 (s, 3H), 1.47 (d, J=7.1 Hz, 3H).

Example 5. General Synthesis of Compound 108 and Related Analogs Step 1: Synthesis of 2-chloro-6-methyl-N-(5-methyl-1H-pyrazol-3-yl)pyrimidin-4-amine

A suspension of 2,4-dichloro-6-methyl-pyrimidine (120.00 g, 736.2 mmol, 1.00 eq), 5-methyl-1H-pyrazol-3-amine (78.65 g, 0.81 mol, 1.10 eq) and DIPEA (142.72 g, 1.10 mol, 1.50 eq) in DMSO (400 mL) was heated at 60° C. for 16 hrs. TLC (PE/EA, 5:1, 1:1) showed the reaction was complete. The reaction mixture was cooled to 30° C. and poured into ice-water (800 mL). The resulting mixture was extracted with MTBE (800 mL×10). The combined organic layers were washed with water (400 mL×3), brine (400 mL×3) and dried over Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure and the residue was recrystallized from DCM (10 mL/g) to afford 2-chloro-6-methyl-N-(5-methyl-1H-pyrazol-3-yl)pyrimidin-4-amine (105.60 g, 472.14 mmol, 64%) as a yellow solid. The structure was confirmed by LC-MS and NMR.

Step 2: Synthesis of methyl 1-methyl-3-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclobutane-1-carboxylate

Methyl 3-iodo-1-methylcyclobutane-1-carboxylate (659 mg, 2.59 mmol) was dissolved in dimethylacetamide (3.2 mL) in a microwave reaction vial. The vial was evacuated and backfilled with nitrogen. Rieke Zinc (4.2 mL of a 50 mg/mL suspension in THF, 3.2 mmol) was added quickly via syringe. The vessel was capped and stirred at ambient temperature for 15 minutes. The vessel was opened under a stream of nitrogen and 2-chloro-6-methyl-N-(5-methyl-1H-pyrazol-3-yl)pyrimidin-4-amine (290 mg, 1.30 mmol) was added followed by PdCl₂dppf (285 mg, 0.389 mmol). The vessel was capped and heated to 80° C. for 2 h. The reaction mixture was then cooled to room temperature, diluted with ethyl acetate, and filtered through celite. The filtrate was transferred to a separatory funnel and washed with water (3×), brine, and dried over sodium sulfate. The dried solution was filtered, and the filtrate was concentrated. The residue was purified by flash-column chromatography on silica gel (gradient elution, 0 to 10% methanol-ethyl acetate) to give methyl 1-methyl-3-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclobutane-1-carboxylate (80 mg, 20%) as a light brown solid. The mixture of diastereomers was not separated at this point. MS (ES+) C₁₆H₂₁N₅O₂ requires: 315, found: 316 [M+H]⁺.

Steps 3 and 4: Synthesis of (1R,3S)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methyl-3-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclobutane-1-carboxamide (Compound 108) and (1S,3R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methyl-3-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclobutane-1-carboxamide (Compound 107)

The title compounds were prepared from methyl 1-methyl-3-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclobutane-1-carboxylate (82 mg, 0.26 mmol) using the same two-step procedure (hydrolysis and amide coupling) as in Synthetic Protocol 1. The diastereomers were separated by reverse-phase HPLC (gradient elution, 10% to 50% acetonitrile-water with 0.1% TFA additive). The fractions containing the compounds were combined and partitioned between ethyl acetate and saturated aqueous sodium bicarbonate solution. The organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated. Peak 1 was Compound 107 (white solid, 5 mg, 4% over two steps). Peak 2 was Compound 108 (white solid, 23 mg, 18% over two steps). The spectral data for these two compounds is presented in the compound table.

Example 6. Synthesis of Ketone Intermediates Ethyl 1-methoxy-3-oxocyclobutane-1-carboxylate

Step 1: Synthesis of 3-(benzyloxy)-1-methoxycyclobutane-1-carboxylic acid

A solution of KOH (12.74 g, 227.0 mmol) in MeOH (70 mL) was added to mixture of 3-(benzyloxy)cyclobutan-1-one (5.00 g, 28.4 mmol) and bromoform (57.4 g, 227 mmol) was added at 0° C. The cooling bath was removed and the mixture was stirred at 20° C. for 16 h. LCMS showed reaction was completed and major was desired product. Water (100 mL) was then added and the mixture was and extracted with DCM (3×80 mL). The aqueous phase was adjusted pH to 3 with aqueous hydrochloric acid solution (0.5 N), then extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine (3×50 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. Crude product (4.8 g) was obtained as brown solid which was used to next step directly without further purification

Step 2: Synthesis of ethyl 3-(benzyloxy)-1-methoxycyclobutane-1-carboxylate

Potassium carbonate (5.62 g, 40.6 mmol) and iodoethane (4.75 g, 30.5 mmol) were added to a solution of 3-(benzyloxy)-1-methoxycyclobutane-1-carboxylic acid (4.80 g, 20.3 mmol) in DMF (40 mL) at 0° C. The reaction mixture was stirred at 20° C. for 16 h, then was partitioned between water and ethyl acetate. The aqueous layer was further extracted with ethyl acetate (2×), and the organic layers were combined. LCMS showed reaction was completed. The mixture was added H₂O (100 mL) and extracted with EA (100 mL*3). The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (PE:EA=10:1) to give ethyl 3-(benzyloxy)-1-methoxycyclobutane-1-carboxylate (4.80 g, 79% yield) as a yellow oil.

Step 3: Synthesis of ethyl 3-hydroxy-1-methoxycyclobutane-1-carboxylate

Palladium hydroxide on carbon (200 mg) was added to a solution of ethyl 3-(benzyloxy)-1-methoxycyclobutane-1-carboxylate (2.0 g, 7.6 mmol) in EtOH (80 mL) at ambient temperature in a high pressure reaction vessel. The vessel was evacuated and backfilled with hydrogen for three cycles, and was then charged with hydrogen at 50 psi. The reaction mixture was stirred for 16 h at 50 psi, then was evacuated and backfilled with nitrogen. The reaction mixture was filtered, and the filtrate was concentrated to give ethyl 3-hydroxy-1-methoxycyclobutane-1-carboxylate (1.0 g, 76% yield) as a light yellow oil. ¹H-NMR (400 MHz, CDCl₃) δ 4.46-4.42 (m, 1H), 4.27 (q, J=7.2 Hz, 2H), 3.27 (s, 3H), 2.66-2.58 (m, 2H), 2.41-2.34 (m, 2H), 1.33 (t, J=7.2 Hz, 3H).

Step 4: Synthesis of ethyl 1-methoxy-3-oxocyclobutane-1-carboxylate

DMP (6.04 g, 14.2 mmol) was added to a solution of ethyl 3-hydroxy-1-methoxycyclobutane-1-carboxylate (1.60 g, 9.19 mmol) in DCM (10 mL) at 20° C. The reaction mixture was stirred for 16 h, and TLC analysis showed formation of a new spot (PE:EA=10:1, Rf=0.35, stained by I₂). The reaction mixture was filtered and the organic phase was concentrated to give crude under reduced pressure. The residue was purified by flash column chromatography on silica gel (PE:EA=10:1) to give the title compound (1.40 g, 75.2% yield) as a yellow oil.

Example 7. Synthesis of Iodide Intermediates Ethyl 3-iodo-1-methoxycyclobutane-1-carboxylate

Step 1: Synthesis of ethyl 1-methoxy-3-(((trifluoromethyl)sulfonyl)oxy)cyclobutane-1-carboxylate

A solution of ethyl 3-hydroxy-1-methoxycyclobutane-1-carboxylate (29.4 g, 129 mmol) and 2,6-lutidine (29.5 mL, 253 mmol) in DCM (500 mL) was cooled to −10° C. Trifluoromethanesulfonic anhydride (50 g, 177 mmol) was then added dropwise over 45 min to the cooled solution. The reaction mixture was stirred for 1 h, then was quenched with saturated ammonium chloride solution (500 mL) and the layers were partitioned. The organic layer was dried over sodium sulfate, filtered, and the filtrate was concentrated to give ethyl 1-methoxy-3-(((trifluoromethyl)sulfonyl)oxy)cyclobutane-1-carboxylate as a brown oil that was used directly in the next step without any purification.

Step 2: Synthesis of ethyl 3-iodo-1-methoxycyclobutane-1-carboxylate

A solution of the entire crude ethyl 1-methoxy-3-(((trifluoromethyl)sulfonyl)oxy)cyclobutane-1-carboxylate from above (assumed to be 169 mmol) in DMF (170 mL) put in a water bath at ambient temperature. Sodium iodide (76 g, 507 mmol) was added to the solution and the reaction mixture was stirred at ambient temperature for 16 h. The reaction mixture was diluted with water (1 L) and extracted with ethyl acetate (2×400 mL). The organic layers were combined and washed sequentially with water (3×1 L), saturated sodium thiosulfate solution, and brine. The organic layer was then dried over sodium sulfate and filtered, and the filtrate was concentrated. The residue was purified by flash-column chromatography on silica gel (gradient elution, 0 to 20% ethyl acetate-hexanes) to give the title compound (31.5 g, 66% yield over two steps) as a colorless oil. MS (ES+) C₈H₁₃IO₃ requires: 284, found: 285 [M+H]⁺. ¹H NMR (400 MHz, Chloroform-d) δ 4.33 (p, J=8.4 Hz, 1H), 4.25 (q, J=7.1 Hz, 2H), 3.24 (s, 3H), 3.20-3.13 (m, 2H), 2.82-2.71 (m, 2H), 1.31 (t, J=7.1 Hz, 3H).

Methyl 3-iodo-1-methylcyclobutane-1-carboxylate

Step 1: Synthesis of methyl 1-methyl-3-((methylsulfonyl)oxy)cyclobutane-1-carboxylate

Methanesulfonyl chloride (0.60 mL, 7.6 mmol) was added to a solution of methyl 3-hydroxy-1-methylcyclobutane-1-carboxylate (1.00 g, 6.94 mmol) and triethylamine (1.26 mL, 9.02 mmol) at 0° C. After the addition, the cooling bath was removed and the reaction mixture was stirred for 70 min. The reaction mixture was then partitioned between DCM and aqueous hydrochloric acid solution (1 N). The organic layer was washed with saturated aqueous sodium bicarbonate solution, brine, and then was dried over sodium sulfate. The mixture was then filtered, and the filtrate was concentrated to give methyl 1-methyl-3-((methylsulfonyl)oxy)cyclobutane-1-carboxylate (1.48 g, 96%) as a light yellow oil that was used in the next step without any further purification.

Step 2: Synthesis of methyl 3-iodo-1-methylcyclobutane-1-carboxylate

Sodium iodide (3.79 g, 25.3 mmol) was added to a solution of methyl 1-methyl-3-((methylsulfonyl)oxy)cyclobutane-1-carboxylate (1.41 g, 6.32 mmol) in DMF (12.6 mL). The reaction mixture was heated to 80° C. for 46 h, then was cooled to ambient temperature and partitioned between DCM and water. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash-column chromatography on silica get (gradient elution, 0 to 15% ethyl acetate-hexanes) to give the title compound (659 mg, 41%) as a colorless oil. MS (ES+) C₇H₁₁IO₂ requires: 254, found: 255 [M+H]⁺.

Example 8. Synthesis of Amine Intermediates (S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethan-1-amine

Step 1: 1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethan-1-one

4-Fluoro-1H-pyrazole (4.73 g, 55 mmol) and potassium carbonate (17.27 g, 125 mmol) were combined and stirred in N,N-dimethylformamide (41.7 mL) for 10 minutes in an open sealed tube before addition of 2-bromo-5-acetylpyridine (10 g, 50 mmol). The reaction tube was sealed and stirred 20 hours at 100° C. The reaction mixture was then cooled to room temperature and poured into water (˜700 mL). The mixture was sonicated and stirred for 20 minutes. A beige solid was isolated by filtration, washed with small amounts of water, and dried to yield 1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethan-1-one (9.81 g, 96% yield). MS: M+1=206.0.

Step 2: (R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-2-methylpropane-2-sulfinamide

To a stirred room temperature solution of 1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethan-1-one (9.806 g, 47.8 mmol) in THF (96 mL) was added (R)-(−)-t-Butylsulfinamide (5.79 g, 47.8 mmol) followed by titanium (IV) ethoxide (21.8 g, 96 mmol). The solution was stirred at 75° C. on an oil bath for 15 hours. The reaction solution was cooled to room temperature and then to −78° C. (external temperature) before the next step. To the −78° C. solution was added dropwise over nearly 55 minutes L-Selectride (143 mL of 1N in THF, 143 mmol). During addition, some bubbling was observed. The reaction was then stirred after the addition was completed for 15 minutes at −78° C. before warming to room temperature. LC-MS of sample taken during removal from cold bath showed reaction was completed. The reaction was cooled to −50° C. and quenched slowly with methanol (˜10 mL), then poured into water (600 mL) and stirred. An off-white precipitate was removed by filtration, with ethyl acetate used for washes. The filtrate was diluted with ethyl acetate (800 mL), the layers were separated, and the organic layer was dried over sodium sulfate, filtered, and concentrated down. The crude was purified by silica gel chromatography to yield (R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-2-methylpropane-2-sulfinamide (10.5 g, 99% purity, 70.3% yield) as a light yellow solid. MS: M+1=311.1.

Step 3: (S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethan-1-amine

A solution of (R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-2-methylpropane-2-sulfinamide (10.53 g, 33.9 mmol)) in methanol (79 mmol) and 4N HCl/dioxane (85 mL, 339 mmol) was stirred 2.5 hours. LC-MS showed reaction was completed. The reaction solution was poured into diethyl ether (300 mL). A sticky solid was formed. The mixture was treated with ethyl acetate (200 mL) and sonicated. The solvents were decanted, and the sticky solid was treated with more ethyl acetate (˜200 mL), sonicated and stirred. The bulk of the sticky solid was converted to a suspension. A light yellow solid was isolated by filtration, washed with smaller amounts of ethyl acetate, and dried to yield (S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethan-1-amine (7.419 g, 78% yield). LC-MS confirmed desired product in high purity. MS: M+1=207.1.

(S)-1-(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)ethan-1-amine

Step 1: 1-(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)ethan-1-one

Sodium hydride (60 wt %, 276 mg, 6.90 mmol) was added to a mixture of 1-(5-chloropyrazin-2-yl)ethanone (800 mg, 5.11 mmol) and 4-Fluoro-1H-pyrazole (484 mg, 5.62 mmol) in N,N-dimethylformamide (6.0 mL) at ambient temperature for 10 minutes. The reaction mixture was then poured into water (70 mL) and was sonicated and stirred for 20 minutes. A dark red solid was isolated by filtration, washed with small amounts of water, and dried to 1-(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)ethan-1-one (919 mg, 95% yield). MS: M+1=207.

Step 2: (R)—N—((S)-1-(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)ethyl)-2-methylpropane-2-sulfinamide

To a stirred room temperature solution of 1-(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)ethan-1-one (4.67 g, 22.7 mmol) in THF (45 mL) was added (R)-(−)-t-Butylsulfinamide (2.75 g, 22.7 mmol) followed by titanium (IV) ethoxide (10.3 g, 45.3 mmol). The solution was stirred at 75° C. on an oil bath for 20 hours. The reaction solution was cooled to room temperature and then to −78° C. before the next step. To the −78° C. solution was added dropwise over 50 minutes L-Selectride (50.1 mL of 1 N in THF, 50.1 mmol). During addition, some bubbling was observed. The reaction was then stirred after the addition was completed for 15 minutes at −78° C. before warming to room temperature. LC-MS of sample taken during removal from cold bath showed reaction was completed. The reaction was cooled to −60° C. and quenched slowly with methanol (1 mL), then poured into water (100 mL) and stirred. The mixture was filtered and the solids were washed further with ethyl acetate. The filtrate was diluted with ethyl acetate, the layers were separated, and the organic layer was dried over sodium sulfate. The dried solution was filtered, and the filtrate was concentrated. The residue was purified by flash-column chromatography (gradient elution, 0 to 100% ethyl acetate-dichloromethane) to give (R)—N—((S)-1-(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)ethyl)-2-methylpropane-2-sulfinamide (1.04 g, 14%) as a brown solid. MS: M+1=312. ¹H NMR (400 MHz, DMSO-d6) δ 9.12 (d, J=1.4 Hz, 1H), 8.73 (d, J=4.5 Hz, 1H), 8.59 (d, J=1.4 Hz, 1H), 8.03 (d, J=4.1 Hz, 1H), 5.69 (d, J=5.7 Hz, 1H), 4.62 (p, J=6.8 Hz, 3H), 1.57 (d, J=6.9 Hz, 3H), 1.12 (s, 9H).

Step 3: (S)-1-(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)ethan-1-amine

A solution of (R)—N—((S)-1-(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)ethyl)-2-methylpropane-2-sulfinamide (1.04 g, 3.34 mmol) in methanol (7.8 mL) and 4N HCl/dioxane (8.34 mL, 33.4 mmol) was stirred for 1.5 h at ambient temperature. The reaction mixture was poured into diethyl ether (100 mL). A light beige solid was isolated by filtration to afford (S)-1-(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)ethan-1-amine (689 mg, 85% yield). MS: M+1=208.

(5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)methanamine

Step 1: 5-(4-fluoro-1H-pyrazol-1-yl)pyrazine-2-carbonitrile

To a solution of 5-chloropyrazine-2-carbonitrile (280 mg, 2.0 mmol) in DMF was added 4-fluoro-1H-pyrazole (170 mg, 2.0 mmol), and potassium acetate (395 mg, 4.0 mmol). The mixture was stirred at the 100° C. for 4 hours. The reaction mixture was cooled to 20° C., poured into brine (25 mL), and extracted with ethyl acetate. The organic layer was dried over sodium sulfate, concentrated and purified by column chromatography (hexane:ethyl acetate=5:1) to give 5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-carbonitrile (310 mg, Yield 82%). The structure was confirmed by LC-MS.

Step 2: (5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)methanamine

A mixture of 5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-carbonitrile (190 mg, 1.0 mmol) and NiCl₂ (12 mg, 0.1 mmol) in MeOH (5 mL) was added NaBH₄ (380 mg, 10 mmol) at 0° C. The mixture was stirred at 0° C. for 2 hours, quenched with aqueous NH₄Cl and purified by HPLC to give (5-(4-fluoro-1H-pyrazol-1-yl)pyrazin-2-yl)methanamine (160 mg, Yield 82%). The structure was confirmed by LC-MS.

(6-(3,5-dimethyl-1H-pyrazol-1-yl)pyridin-3-yl)methanamine

Step 1: 6-(3,5-Dimethyl-1H-pyrazol-1-yl)nicotinonitrile

To the solution of 6-chloronicotinonitrile (300 mg, 2.2 mmol) in DMF (10 mL), was added 3,5-dimethyl-1H-pyrazole (210 mg, 2.2 mmol) and Cs₂CO₃ (1.4 g, 4.4 mmol). The mixture was stirred at 90° C. for 16 h. Water (25 mL) was added to the reaction mixture, and the mixture was filtered. The solids were washed with water and dried under vacuum to give 6-(3,5-Dimethyl-1H-pyrazol-1-yl)nicotinonitrile (320 mg, yield 74.6%).

Step 2: tert-Butyl ((6-(3,5-dimethyl-1H-pyrazol-1-yl)pyridin-3-yl)methyl)carbamate

To 6-(3,5-Dimethyl-1H-pyrazol-1-yl)nicotinonitrile (300 mg, 1.5 mmol) in MeOH (10 mL), was added NiCl₂ (19 mg, 0.15 mmol), (Boc)₂O (654 mg, 3.0 mmol) and NaBH₄ (142 mg, 3.8 mmol). The mixture was stirred at ambient temperature for 3 h. Saturated aqueous ammonium chloride solution was added and the MeOH was removed under vacuum. The aqueous suspension was then partitioned with ethyl acetate, and the layers were separated. The organic layer was washed with saturated sodium bicarbonate solution (2×50 mL). The organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated under vacuum to give 450 mg target compound which was used in the next step without further purification.

Step 3: 6-(3,5-Dimethyl-1H-pyrazol-1-yl)nicotinonitrile

A solution of HCl in Dioxane (4.0 M, 10 mL) was added to compound tert-Butyl ((6-(3,5-dimethyl-1H-pyrazol-1-yl)pyridin-3-yl)methyl)carbamate (450 mg). The mixture was stirred for 2 h, then was dried under reduced pressure to give the title compound (350 mg) as a light brown solid that was used without further purification. ¹H NMR (400 MHz, DMSO-d6) δ 8.51 (d, J=2.1 Hz, 1H), 8.34 (s, 3H), 8.03 (dd, J=8.5, 2.4 Hz, 1H), 7.87 (d, J=8.5 Hz, 1H), 6.14 (s, 1H), 4.12 (q, J=5.7 Hz, 2H), 2.59 (s, 3H), 2.21 (s, 3H).

(6-(4-chloro-1H-pyrazol-1-yl)pyridin-3-yl)methanamine

Step 1: 6-(4-Chloro-1H-pyrazol-1-yl)nicotinonitrile

To a solution of 6-chloronicotinonitrile (300 mg, 2.2 mmol) in DMF (10 mL), was added 4-chloro-1H-pyrazole (227 mg, 2.2 mmol) and Cs₂CO₃ (1.4 g, 4.4 mmol). The mixture was stirred at 90° C. for 16 h. Water (25 mL) was added to the mixture, and the mixture was filtered. The solids were washed with water and dried under vacuum to give 6-(4-Chloro-1H-pyrazol-1-yl)nicotinonitrile (380 mg, 84%).

Step 2: tert-Butyl ((6-(4-chloro-1H-pyrazol-1-yl)pyridin-3-yl)methyl)carbamate

To 6-(4-chloro-1H-pyrazol-1-yl)nicotinonitrile (350 mg, 1.7 mmol) in MeOH (10 mL), was added NiCl₂ (19 mg, 0.17 mmol), (Boc)₂O (741 mg, 3.4 mmol) and NaBH₄ (163 mg, 4.3 mmol). The mixture was stirred at ambient temperature. for 3 h. Saturated aqueous ammonium chloride solution was added and the MeOH was removed under vacuum. The aqueous suspension was then partitioned with ethyl acetate, and the layers were separated. The organic layer was washed with saturated sodium bicarbonate solution (2×50 mL). The organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated under vacuum to give 480 mg target compound, which was used in the next step without further purification.

Step 3: (6-(4-chloro-1H-pyrazol-1-yl)pyridin-3-yl)methanamine

A solution of HCl in Dioxane (4.0 M, 10 mL) was added to tert-Butyl ((6-(4-chloro-1H-pyrazol-1-yl)pyridin-3-yl)methyl)carbamate (450 mg, 1.5 mmol) at ambient temperature. The mixture was stirred for 2 h, then was dried under reduced pressure to give the title compound (290 mg) as a light brown solid that was used without further purification. MS: M+1=209.

The synthetic protocols that were used to prepare the compounds disclosed herein are indicated below. The NMR and LC MS data obtained for compounds disclosed herein are also shown below.

TABLE 5 Compound Synthetic MS Number Protocol ¹H NMR (M + 1) 100 2 ¹H NMR (400 MHz, DMSO-d6) δ 11.65 (s, 1H), 8.85 433 (s, 1H), 8.59 (d, J = 2.6 Hz, 1H), 8.42-8.32 (m, 2H), 7.94-7.83 (m, 2H), 7.80 (d, J = 1.6 Hz, 1H), 6.90 (s, 1H), 6.56 (dd, J = 2.6, 1.7 Hz, 1H), 6.40 (s, 1H), 6.15 (s, 1H), 4.35 (d, J = 5.9 Hz, 2H), 3.57-3.43 (m, 1H), 3.15- 3.07 (m, , 1H), 2.44 (d, J = 8.5 Hz, 4H), 2.17 (d, J = 2.6 Hz, 6H 101 2 ¹H NMR (400 MHz, DMSO-d6) δ 11.65 (s, 1H), 8.85 457 (s, 1H), 8.59 (d, J = 2.6 Hz, 1H), 8.40 (d, J = 1.9 Hz, 1H), 8.30 (d, J = 7.8 Hz, 1H), 7.96-7.84 (m, 2H), 7.80 (d, J = 1.6 Hz, 1H), 6.90 (s, 1H), 6.55 (t, J = 2.1 Hz, 1H), 6.38 (s, 1H), 6.16 (s, 1H), 5.09-4.97 (m, 1H), 3.52- 3.39 (m, 1H), 3.17-3.07 (m, 1H), 2.46-2.32 (m, 4H), 2.18 (s, 3H), 2.16 (s, 3H), 1.41 (d, J = 7.0 Hz, 3H). 102 2 ¹H NMR (400 MHz, DMSO-d₆) δ ppm 11.66 (s, 1H), 475 8.87 (s, 1H), 8.67 (d, J = 4.5 Hz, 1H), 8.40 (d, J = 2.2 Hz, 1H), 8.32 (d, J = 7.7 Hz, 1H), 8.00-7.79 (m, 3H), 6.90 (s, 1H), 6.38 (s, 1H), 6.16 (s, 1H), 5.07-4.98 (m, 1H), 3.55-3.40 (m, 1H), 3.16-3.06 (m, 1H), 2.46- 2.31 (m, 4H), 2.17 (s, 3H), 2.16 (s, 3H), 1.40 (d, J = 7.0 Hz, 3H). 103 1 ¹H NMR (400 MHz, DMSO-d6) δ ppm 11.60 (s, 1H), 475 8.85 (s, 1H), 8.65 (d, J = 4.5 Hz, 1H), 8.38 (d, J = 2.2 Hz, 1H), 8.27 (d, J = 7.7 Hz, 1H), 7.95-7.79 (m, 3H), 6.82 (s, 1H), 6.36 (s, 1H), 6.25 (s, 1H), 5.05-4.95 (m, 1H), 3.06-2.95 (m, 1H), 2.47-2.21 (m, 4H), 2.15 (s, 3H), 2.12 (s, 3H), 1.40 (d, J = 7.1 Hz, 3H). 104 2 ¹H NMR (400 MHz, DMSO-d6) δ 11.65 (s, 1H), 9.13 476 (d, J = 1.4 Hz, 1H), 8.85 (s, 1H), 8.72 (dd, J = 4.6, 0.8 Hz, 1H), 8.48 (d, J = 1.4 Hz, 1H), 8.36 (d, J = 7.3 Hz, 1H), 8.02 (d, J = 4.1 Hz, 1H), 6.90 (s, 1H), 6.38 (s, 1H), 6.16 (s, 1H), 513-5.06 (m, 1H), 3.46 (t, J = 7.9 Hz, 1H), 3.18-3.11(m, 1H), 3.00 (td, J = 6.6, 3.9 Hz, 2H), 2.18 (s, 2H), 2.16 (s, 3H), 1.77-1.67 (m, 2H), 1.44 (d, J = 7.1 Hz, 3H). 105 3 ¹H NMR (400 MHz, DMSO-d6) δ ppm 11.90 (s, 1H), 476 9.53 (s, 1H), 8.67 (d, J = 4.5 Hz, 1H), 8.39 (d, J = 2.1 Hz, 1H), 8.31 (d, J = 7.7 Hz, 1H), 7.96-7.81 (m, 3H), 6.87 (s, 1H), 6.13 (s, 1H), 5.06-4.97 (m, 1H), 3.50- 3.40 (m, 1H), 3.19-3.09 (m, 1H), 2.49-2.31 (m, 4H), 2.25 (s, 3H), 2.19 (s, 3H), 1.40 (d, J = 7.0 Hz, 3H). 107 3 ¹H NMR (400 MHz, DMSO-d6) δ 11.91 (s, 1H), 9.56 490 (s, 1H), 8.64 (d, J = 4.5 Hz, 1H), 8.38 (d, J = 2.2 Hz, 1H), 8.03 (d, J = 7.9 Hz, 1H), 7.99-7.79 (m, 3H), 6.80 (s, 1H), 6.19 (s, 1H), 5.05-4.97 (m, 1H), 3.47 (p, J = 8.9 Hz, 1H), 2.60 (q, J = 10.1 Hz, 2H), 2.22 (s, 3H), 2.18- 2.02 (m, 5H), 1.53-1.37 (m, 6H). 108 3 ¹H NMR (400 MHz, DMSO-d6) δ ppm 11.92 (s, 1H), 490 9.56 (s, 1H), 8.69 (d, J = 4.5 Hz, 1H), 8.41 (d, J = 2.2 Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 8.00-7.83 (m, 3H), 6.80 (s, 1H), 6.22 (s, 1H), 5.13-4.98 (m, 1H), 3.27- 3.17 (m, 1H), 2.76-2.59 (m, 2H), 2.30-2.12 (m, 8H), 1.45 (d, J = 7.1 Hz, 3H), 1.34 (s, 3H). 110 3 ¹H NMR (400 MHz, DMSO-d6) δ ppm 11.91 (s, 1H), 491 9.55 (s, 1H), 9.13 (d, J = 1.4 Hz, 1H), 8.74 (d, J = 4.4 Hz, 1H), 8.47 (d, J = 1.5 Hz, 1H), 8.24 (d, J = 7.4 Hz, 1H), 8.02 (d, J = 4.2 Hz, 1H), 6.80 (s, 1H), 6.21 (s, 1H), 5.19-5.03 (m, 1H), 3.28-3.18 (m, 1H), 2.78-2.66 (m, 2H), 2.28-2.15 (m, 8H), 1.49 (d, J = 7.1 Hz, 3H), 1.36 (s, 3H). 114 1, ¹H NMR (400 MHz, DMSO-d6) δ 11.73 (s, 1H), 9.05 493 minor (s, 1H), 8.67 (d, J = 4.5 Hz, 1H), 8.49 (d, J = 7.7 Hz, isomer 1H), 8.39 (d, J = 2.1 Hz, 1H), 7.99-7.80 (m, 3H), 7.01 (s, 1H), 6.67 (s, 1H), 6.14 (s, 1H), 5.09-4.93 (m, 1H), 2.92 (p, J = 9.1, 8.6 Hz, 1H), 2.83-2.69 (m, 2H), 2.69- 2.50 (m, 2H), 2.20 (d, J = 17.2 Hz, 6H), 1.39 (d, J = 6.9 Hz, 3H). 116 1 ¹H NMR (400 MHz, DMSO-d6) δ 11.74 (s, 1H), 8.88 505 (s, 1H), 8.67 (d, J = 4.5 Hz, 1H), 8.43-8.25 (m, 1H), 7.98-7.75 (m, 3H), 6.80 (s, 1H), 6.76 (s, 1H), 6.09 (s, 1H), 5.92-5.82 (m, 1H), 5.68 (s, 1H), 3.38-3.26 (m, 1H), 2.70-2.55 (m, 5 H), 2.25-2.05 (m, 8H), (s, 2H), 2.20 (s, 2H), 2.11 (s, 2H), 1.50 (d, J = 7.1 Hz, 3H). 118 1 ¹H NMR (400 MHz, DMSO-d6) δ 12.83 (s, 1H), 11.60 505 (s, 1H), 8.67 (d, J = 4.6 Hz, 1H), 8.38 (d, J = 2.1 Hz, 1H), 8.33 (d, J = 7.7 Hz, 1H), 7.92 (dd, J = 8.1, 2.9 Hz, 2H), 7.86 (d, J = 8.5 Hz, 1H), 6.89 (s, 1H), 6.75 (s, 1H), 5.92 (s, 1H), 3.00 (d, J = 13.1 Hz, 1H), 2.89 (d, J = 13.0 Hz, 1H), 2.34 (d, J = 13.4 Hz, 2H), 2.28 (s, 3H), 2.25 (s, 3H), 1.58 (s, 3H), 1.43 (d, J = 7.1 Hz, 3H). 119 1 ¹H NMR (400 MHz, DMSO-d6) δ ppm 11.68 (s, 1H), 505 8.87 (s, 1H), 8.67 (d, J = 4.5 Hz, 1H), 8.44 (d, J = 2.2 Hz, 1H), 8.18 (d, J = 7.8 Hz, 1H), 8.04-7.94 (m, 1H), 7.94-7.80 (m, 2H), 6.79 (s, 1H), 6.70 (s, 1H), 6.29 (s, 1H), 5.46 (s, 1H), 5.15-4.97 (m, 1H), 2.55 (s, 4H), 2.25-2.12 (m, 6H), 1.45 (d, J = 7.1 Hz, 3H), 1.35 (s, 3H). 120 2 ¹H NMR (400 MHz, DMSO-d6) δ 11.68 (s, 1H), 8.94 505 (s, 1H), 8.67 (d, J = 4.5 Hz, 1H), 8.51 (d, J = 8.3 Hz, 1H), 8.45 (d, J = 2.2 Hz, 1H), 8.00 (dd, J = 8.6, 2.3 Hz, 1H), 7.94-7.85 (m, 2H), 6.80 (s, 1H), 6.40 (s, 1H), 6.27 (s, 1H), 5.21-4.94 (m, 1H), 3.19-3.00 (m, 4H), 2.70-2.35 (m, 4H), 2.17 (d, J = 6.7 Hz, 6H), 1.48 (d, J = 7.0 Hz, 3H). 121 3 ¹H NMR (400 MHz, DMSO-d6) δ 11.93 (s, 1H), 9.61 506 (s, 1H), 8.67 (d, J = 4.5 Hz, 1H), 8.49 (d, J = 8.2 Hz, 1H), 8.44 (d, J = 2.2 Hz, 1H), 8.00 (dd, J = 8.5, 2.3 Hz, 1H), 7.93-7.85 (m, 2H), 6.84 (s, 1H), 6.20 (s, 1H), 5.16-5.01 (m, 1H), 3.10 (s, 4H), 2.69-2.39 (m, 4H), 2.24 (s, 3H), 2.19 (s, 3H), 1.47 (d, J = 7.1 Hz, 3H). 123 3 ¹H NMR (400 MHz, DMSO-d6) δ 11.90 (s, 1H), 9.66- 507 9.48 (m, 1H), 9.13 (d, J = 1.3 Hz, 1H), 8.73 (d, J = 4.5 Hz, 1H), 8.54 (d, J = 1.3 Hz, 1H), 8.41 (d, J = 7.8 Hz, 1H), 8.02 (d, J = 4.1 Hz, 1H), 6.84 (s, 1H), 6.21 (s, 1H), 5.29-5.12 (m, 1H), 3.23-3.08 (m, 4H), 2.70-2.43 (m, 4H), 2.24 (s, 3H), 2.19 (s, 3H), 1.51 (d, J = 7.1 Hz, 3H). 124 2 ¹H NMR (400 MHz, DMSO-d6) δ 11.77 (s, 1H), 9.24 507 (s, 1H), 9.13 (d, J = 1.4 Hz, 1H), 8.73 (d, J = 4.5 Hz, 1H), 8.54 (d, J = 1.4 Hz, 1H), 8.44 (d, J = 7.9 Hz, 1H), 8.02 (d, J = 4.1 Hz, 1H), 6.54 (s, 2H), 5.25-5.06 (m, 1H), 3.17 (s, 4H), 2.71-2.37 (m, 4H), 2.26 (s, 3H), 2.19 (s, 3H), 1.51 (d, J = 7.0 Hz, 3H). 126 1 ¹H NMR (400 MHz, DMSO-d6) δ 10.04-9.91 (m, 507 1H), 8.62 (d, J = 4.4 Hz, 1H), 8.32 (s, 2H), 7.91 (d, J = 4.1 Hz, 1H), 7.84 (s, 1H), 7.79 (d, J = 8.5 Hz, 1H), 6.80 (s, 1H), 6.74 (s, 1H), 6.14 (s, 1H), 5.09-5.04 (m, 1H), 3.28-3.22 (m, 1H), 3.14-3.08 (m, 1H), 2.40-2.36 (m, 1H), 2.25 (s, 3H), 2.21 (s, 3H), 2.04-1.96 (m, 1H), 1.61 (s, 3H). 127 1 ¹H NMR (400 MHz, DMSO-d6) δ 11.67 (s, 1H), 8.99 521 (s, 1H), 8.72 (d, J = 8.1 Hz, 1H), 8.68 (d, J = 4.5 Hz, 1H), 8.46 (d, J = 2.2 Hz, 1H), 8.02 (dd, J = 8.5, 2.3 Hz, 1H), 7.94-7.85 (m, 2H), 6.73 (s, 1H), 6.46 (s, 1H), 5.81 (s, 1H), 5.21-5.08 (m, 1H), 3.16 (s, 3H), 2.93 (t, J = 11.9 Hz, 2H), 2.49 (s, 3H), 2.45-2.34 (m, 2H), 2.18 (s, 3H), 1.50 (d, J = 7.1 Hz, 3H). 128 1 ¹H NMR (400 MHz, DMSO-d6) δ 11.61 (s, 1H), 8.80 521 (s, 1H), 8.63 (d, J = 4.5 Hz, 1H), 8.37 (t, J = 4.1 Hz, 2H), 7.94-7.86 (m, 2H), 7.82 (d, J = 8.5 Hz, 1H), 6.80 (s, 1H), 6.66 (s, 1H), 6.22 (s, 1H), 5.50 (s, 1H), 5.06- 4.99 (m, 1H), 3.14-3.03 (m, 5H), 2.32-2.25 (m, 2H), 2.14 (s, 3H), 2.13 (s, 3H), 1.42 (d, J = 7.0 Hz, 3H). 131 2 ¹H NMR (400 MHz, DMSO-d6) δ 11.77 (s, 1H), 9.24 493 (s, 1H), 9.12 (d, J = 1.4 Hz, 1H), 8.72 (d, J = 4.5 Hz, 1H), 8.65 (t, J = 6.0 Hz, 1H), 8.44 (d, J = 1.4 Hz, 1H), 8.02 (d, J = 4.1 Hz, 1H), 6.55 (s, 2H), 4.53 (d, J = 5.9 Hz, 2H), 3.22-3.14 (m, 4H), 2.67-2.42 (m, 4H), 2.27 (s, 3H), 2.19 (s, 3H).

Example 9: Measurement of Biochemical Activity of Compounds

In order to assess the activity of chemical compounds against the relevant kinase of interest, the Caliper LifeSciences electrophoretic mobility shift technology platform is used. Fluorescently labeled substrate peptide is incubated in the presence of kinase and ATP so that a reflective proportion of the peptide is phosphorylated. At the end of the reaction, the mix of phosphorylated (product) and non-phosphorylated (substrate) peptides are passed through the microfluidic system of the Caliper EZ Reader 2, under an applied potential difference. The presence of the phosphate group on the product peptide provides a difference in mass and charge between those of the substrate peptide, resulting in a separation of the substrate and product pools in the sample. As the pools pass a LEDS within the instrument, these pools are detected and resolved as separate peaks. The ratio between these peaks therefore reflects the activity of the chemical matter at that concentration in that well, under those conditions.

RET Wild Type Assay at KM

In each well of a 384-well plate, 7.5 nM-10 nM of wild type RET (ProQinase 1090-0000-1) is incubated in a total of 12.5 μL of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl₂, 1 mM DTT) with 1 μM CSKtide (FITC-AHA-KKKKD DIYFFFG-NH2) (SEQ ID NO: 5) and 25 μM ATP at 25° C. for 120 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction is stopped by the addition of 70 μL of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate is then read on a Caliper EZReader 2 (protocol settings: −1.7 psi, upstream voltage −500, downstream voltage −3000, post sample sip 35s). Data is normalized to 0% and 100% inhibition controls and the IC₅₀ calculated using a 4-parameter fit in the CORE LIMS.

RET V804L Gatekeeper Mutant Assay at KM

In each well of a 384-well plate, 7.5 nM-10 nM of mutant RET (ProQinase 1096-0000-1) is incubated in a total of 12.5 μL of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 μM CSKtide (FITC-AHA-KKKKDDIYFFFG-NH2) (SEQ ID NO: 5) and 10 μM ATP at 25° C. for 120 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction is stopped by the addition of 70 μL of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate is then read on a Caliper EZReader 2 (protocol settings: −1.7 psi, upstream voltage −500, downstream voltage −3000, post sample sip 35s). Data is normalized to 0% and 100% inhibition controls and the IC₅₀ calculated using a 4-parameter fit in the CORE LIMS.

In Table 6 below, the following designations are used: ≦1.00 nM=A; 1.01-10 nM=B; 10.01-100 nM=C; <100 nM=D; and ND=not determined.

TABLE 6 RET Inhibitory Activity of Exemplary Compounds of the Invention. Wild-type Compound RET V804L Mutant 100 A A 101 A A 102 A A 103 C B 104 A A 105 A A 107 B B 108 A A 109 A A 110 A A 113 C C 114 B B 116 B B 117 D C 118 D D 119 A A 120 A A 121 A A 122 A A 123 A A 124 A A 126 D D 127 A A 128 D D 131 B ND

Example 10: Compounds of Structural Formula (I) are Potent Inhibitors of Wild-Type and Mutant RET

In embodiments, compounds according to structural Formula (I) and structural Formula (II) are potent and selective inhibitors of oncogenic RET mutant and fusion proteins. Currently, there are no approved inhibitors that selectively target these disease-driving RET alterations.

1. In Vitro Assays

A compound described herein can be tested in vitro for inhibition of wild-type RET and various mutant RET kinases, including e.g., RET V804L, RET V804M, and RET M918T kinases, as well as CCDC6-RET and KIF5B-RET fusion kinases. The IC50 can be calculated.

2. Cellular Assays

In cellular systems, the activity of a compound of structural Formula (I) or structural Formula (II) can be measured by inhibition of RET mutant or RET fusion autophosphorylation, RET-dependent signaling, and by inhibition of RET dependent cell proliferation. The compound can be assayed for activity in cancer cell lines endogenously expressing activated RET fusions or mutants. Exemplary cells that can be used for these studies include patient xenografts and established cell lines. Exemplary patient xenografts include Colorectal Cancer patient derived xenograft, Lung adenocarcinoma patient derived xenograft, and NSCLC patient derived xenograft. Exemplary cell lines include Ba/F3-KIF5B-RET (a model for leukemia), LC2/ad cells (a model for lung cancer), MZ-CRC 1 (a model for thyroid cancer), and TT cells (a medullary thyroid cancer cell line). Exemplary RET mutations that can be used for these studies include fusions such as KIF5B-RET and CCDC6-RET; point mutations such as RET C634W, RET V804L, RET V804E, RET V804M, and RET M918T; and fusions containing point mutations such as KIF5B-RET (V804L) and KIF5B-RET (V804M). KIF5B-RET (V804L) refers to a mutant RET that comprises a fusion with KIF5B and further comprises a V804L mutation in RET (referring the amino acid numbering of wild-type RET). KIF5B-RET (V804M) refers to a mutant RET that comprises a fusion with KIF5B and further comprises a V804M mutation in RET.

2a. Autophosphorylation Assays

As an example of an autophosphorylation assay, in Ba/F3 models engineered to express KIF5B RET, the compound is tested for its ability to inhibit RET fusion protein signaling as measured by inhibition of RET autophosphorylation. IC50 can be calculated. Wild-type RET inhibitors cabozantinib and vandetanib can be used as controls (less potent compounds than the test compound) in these cellular assays. In embodiments, the compound of structural Formula (I) and (II) potently and selectively inhibit RET autophosphorylation.

The compound can also be tested for ability to inhibit RET autophosphorylation in LC2/ad cells, a non-engineered NSCLC cell line that expressed a CCDC6-RET fusion (Suzuki et al, 2013). For instance, immunoblot is performed for LC2/ad cells expressing the CCDC6-RET fusion protein and treated with the test compound at different concentrations; Phosphorylated (phospho[Y1062]) and total protein levels of RET are measured.

2b. Proliferation Assays

In embodiments, the compound of structural Formula (I) and (II) inhibits proliferation.

In proliferation assays, the compound can be tested for ability to inhibit KIF5B-RET dependent Ba/F3 cell growth. The IC50 can be calculated.

Inhibition of RET activity with the compound can also be tested for inhibition of proliferation of the CCDC6-RET expressing cell line. Similarly, the compound can also be tested for its ability to inhibit RET pathway signaling and RET dependent proliferation in the human MTC TT and MZ-CRC 1 cell lines, driven by RET C634W or RET M918T mutations, respectively. In some embodiments, in RET-driven cell lines, the test compound inhibits RET activity and RET-driven proliferation more potently than the multi-kinase inhibitors such as cabozantinib and vandetanib.

2c. Downstream Signalling Assays

In LC2/ad cells, a non-engineered NSCLC cell line that expressed a CCDC6-RET fusion (Suzuki et al, 2013), the compound can be tested for its ability to inhibit phosphorylation of the RET substrate Src homology domain (Shc) (Hayashi et al, 2000), and downstream signaling through extracellular signal regulated kinase (ERK)1/2, including downregulation of dual specificity phosphatase 6 (DUSP6) and sprouty receptor tyrosine kinase signaling antagonist 4 (SPRY4) (Lito et al, 2013). For instance, immunoblot can be performed for LC2/ad cells expressing the CCDC6-RET fusion protein and treated with the test compound at different concentrations; phosphorylated and total levels of downstream biomarkers, e.g., phospho(Y239/Y240)-Shc and phospho(Y202/T204)-ERK1/2, are measured. In addition, to determine expression levels of downstream targets, LC2/ad cells can be treated with the compound, cabozantinib, or DMSO for 7 hours and RNA is harvested. Gene expression levels of DUSP6 and SPRY4 can be measured by qRT-PCR. In embodiments, the compound induces a dose-dependent decrease in expression of the ERK1/2 target genes DUSP6 and SPRY4 but not the control gene glycogen synthase kinase 3 beta (GSK3B).

3. Animal Models

Antitumor efficacy of compounds of structural Formula (I) and (II) can be demonstrated in several RET-driven in vivo models. The Ba/F3-KIF5B-RET allograft model uses KIF5B RET fusion-dependent Ba/F3 cells. The test compound can be administered orally with an appropriate dose. Tumor size can be measured e.g., twice weekly. In embodiments, administration of the compound results in robust and dose-dependent growth inhibition of Ba/F3-KIF5B-RET allograft tumors, e.g., in complete TGI and Mouse body weight can be measured e.g., twice-weekly during the administration. In embodiments, the compound is well tolerated with no significant changes in animal body weight observed.

Similar assays can be performed using other animal models, including a Ba/F3-KIF5B-RET (V804L) allograft tumor model which comprises a KIF5B RET V804L fusion protein, an KIF5B-RET NSCLC allograft tumor model, an MTC cell line xenograft driven by a RET C634W mutation, and a CCDC6-RET fusion positive colorectal cancer allograft tumor model. The RET V804L mutation has been observed in rare cases of MTC and is predicted to be insensitive to cabozantinib and vandetanib in vitro and in vivo (Carlomagno et al, 2004; Bentzien et al, 2013; BPM 0016). In embodiments, the compound causes complete TGI and regressions in a cancer that is not responsive to cabozantinib or vandetanib.

Biochemical markers can also be assayed in the treated mice. To assess direct inhibition of KIF5B-RET (V804L) fusion kinase activity in Ba/F3 KIF5B-RET (V804L) tumors, the compound can be administered orally at an appropriate dose to tumor bearing mice for several days and plasma and tumors can be collected from individual mice at appropriate time points after the last dose. Test compound concentrations in plasma can be determined by liquid chromatograph/tandem mass spectrometry (LC/MS/MS). Inhibition of KIF5B-RET (V804L) signaling in the tumor tissue can be assessed by a phosphor RET enzyme linked immunosorbent assay (ELISA) and by immunoblotting, e.g., as described above. Quantitation of the phospho-RET signal by ELISA can measure the percent KIF5B-RET (V804L) inhibition in treated animals as compared to vehicle treated controls. Suppression of downstream RET pathway signaling can be demonstrated by inhibition of Shc phosphorylation. In embodiments, a dose and time-dependent correlation is observed between the concentration of the test compound in mouse plasma and the level of phosphorylated KIF5B RET (V804L). In embodiments, administration of the compound at an amount sufficient to reach at least 90% inhibition of RET in vivo leads to therapeutic efficacy, e.g., can lead to 100% tumor growth inhibition.

Example 11: Selectivity of Compounds of Structural Formulas (I) and (II) Efficacy Against Wild-Type and Mutant RET

In embodiments, compounds according to structural Formula (I) and structural Formula (II) are potent inhibitors of wild-type and mutant RET. For instance the IC50 of a compound can be tested in a cell line comprising wild-type RET and in a second cell line comprising mutant RET, e.g., a point mutation or fusion.

Selectivity for RET Over KDR

In embodiments, compounds according to structural Formula (I) and structural Formula (II) are selective for RET over another kinase, such as KDR (also called Vascular endothelial growth factor receptor 2). KDR is a tyrosine-protein kinase that acts as a cell-surface receptor for VEGFA, VEGFC and VEGFD. Inhibition of KDR/VEGFR2 has been associated clinically with certain adverse effects, e.g., hypertension, arterial thrombosis, and hemorrhage, and therefore selectivity for RET over KDR is desirable.

To test selectivity, the test compound can be assayed for its ability to inhibit proliferation in parental Ba/F3 cells that do not express a RET mutation, e.g., does not express a KIF5B-RET fusion. A weak IC50 in the parental cell line indicates that the test compound is selective for cell lines dependent on oncogenic RET.

The selectivity of a compound on RET versus other human kinases can be characterized by profiling binding across a panel of over 450 human kinases and disease-relevant kinase mutants. In embodiments, the compound has a high degree of selectivity for RET and RET kinase mutants over other kinases tested. To define the binding affinity for the kinases bound by the compound in kinome screening and additional kinases of interest, the dissociation constant (Kd) can be determined.

To differentiate the compound from multi-kinase inhibitors with biochemical activity against RET, the activity of the compound against recombinant kinase insert domain receptor (KDR) (also known as vascular endothelial growth factor receptor 2 [VEGFR2]) and fibroblast growth factor receptor 1 (FGFR1) can be tested, as inhibition of these kinases is associated with dose-limiting toxicities in humans. Inhibition of KDR/VEGFR2 has been associated clinically with hypertension, arterial thrombosis, and hemorrhage, whereas inhibition of fibroblast growth factor receptors (FGFRs) is associated with hyperphosphatemia and tissue calcification (Abdel-Rahman and Fouad, 2014; Touat et al, 2015). In embodiments, the compound is a more potent inhibitor of WT RET than KDR/VEGFR2 and FGFR1, respectively. In contrast, in embodiments a multi-kinase inhibitor exhibits approximately equal or increased potency on KDR versus WT RET.

Example 12: Selective Compounds Prevent RET Resistance Mutants

The compounds herein can be tested for the propensity of a cancer to develop one or more RET mutations associated with drug resistance. For example, RET-altered cancer cells (e.g., Ba/F3 KIF5B-RET cells) can be treated with a mutagen such as ENU, exposed to a compound herein or a control compound (e.g., for 2-3 weeks), and the cell number can be quantified. Cells with high proliferation can be subjected to DNA sequencing to detect RET mutations. In embodiments, a compound of structural Formula (I) or (II) leads to no or fewer RET mutations than a control compound such as a multi-kinase inhibitor such as cabozantinib.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. 

1. A compound having the structural Formula (I):

or a pharmaceutically acceptable salt thereof, wherein: ring A is an aryl or heteroaryl ring; each of X¹ and X² is independently selected from N and C(R⁶); each R¹ and each R⁷ is independently selected from selected from C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxy, halo, C₁-C₆ heteroalkyl, cycloalkyl, aryl, heteroaryl, aryloxy, aralkyl, heterocyclyl, heterocyclylalkyl, nitro, cyano, —C(O)R, —OC(O)R, —C(O)OR, —(C₁-C₆ alkylene)-C(O)R, —SR, —S(O)₂R, —S(O)₂—N(R)(R), —(C₁-C₆ alkylene)-S(O)₂R, —(C₁-C₆ alkylene)-S(O)₂—N(R)(R), —N(R)(R), —C(O)—N(R)(R), —N(R)—C(O)R, —N(R)—C(O)OR, —(C₁-C₆ alkylene)-N(R)—C(O)R, —N(R)S(O)₂R, and —P(O)(R)(R); wherein each of alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, cycloalkyl, aryl, heteroaryl, aryloxy, aralkyl, heterocyclyl, and heterocyclylalkyl is independently substituted with 0-5 occurrences of R^(a); or two R¹ or two R⁷ are taken together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclyl ring independently substituted with 0-5 occurrences of R^(b); each of R², R^(3a), R^(3b), R⁴, R^(8a) and R^(8b) is independently selected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, halo, hydroxyl, C₁-C₆ heteroalkyl, and —N(R)(R); wherein each alkyl, alkoxy, and heteroalkyl is optionally and independently substituted with 0-5 occurrences of R^(a); each of R⁵ and R⁹ is independently selected from hydrogen, C₁-C₆ alkyl, and C₁-C₆ heteroalkyl; wherein each alkyl and heteroalkyl is optionally and independently substituted with 0-5 occurrences of R^(a); each R⁶ is independently selected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, halo, C₁-C₆ heteroalkyl, and —N(R)(R); wherein each alkyl, alkoxy, and heteroalkyl is optionally and independently substituted with 0-5 occurrences of R^(a); each R is independently selected from hydrogen, hydroxyl, halo, thiol, C₁-C₆ alkyl, C₁-C₆ thioalkyl, C₁-C₆ alkoxy, C₁-C₆ heteroalkyl, cycloalkyl, cycloalkylalkyl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl, wherein each of alkyl, thioalkyl, alkoxy, heteroalkyl, cycloalkyl, cycloalkylalkyl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl is independently substituted with 0-5 occurrences of R^(a), or two R together with the atom(s) to which they are attached form a cycloalkyl or heterocyclyl ring independently substituted with 0-5 occurrences of R^(b); each R^(a) and each R^(b) is independently selected from C₁-C₆ alkyl, halo, hydroxyl, C₁-C₆ heteroalkyl, C₁-C₆ alkoxy, cycloalkyl, heterocyclyl, or cyano, wherein each of alkyl, heteroalkyl, alkoxy, cycloalkyl and heterocyclyl is independently substituted with 0-5 occurrences of R′; each R′ is independently selected from C₁-C₆ alkyl, C₁-C₆ heteroalkyl, halo, hydroxyl, cycloalkyl or cyano; or two R′ together with the atom(s) to which they are attached form a cycloalkyl or heterocyclyl ring; m is 0, 1, or 2; and n is 0, 1, 2, or
 3. 2. The compound of claim 1, wherein m is 1; R¹ is located at the 5-position; and R¹ is C₁-C₄ alkyl substituted with 0-3 occurrences of R^(a).
 3. The compound of claim 2, wherein R¹ is —CH₃.
 4. The compound of any of claims 1-3, wherein R² is selected from hydrogen, hydroxyl, halo and O—C₁-C₄ alkyl.
 5. The compound of claim 4, wherein R² is selected from hydrogen, hydroxyl, fluoro and —OCH₃.
 6. The compound of any one of claims 1-5, wherein each of R^(3a), R^(3b), R^(8a) and R^(8b) is hydrogen.
 7. The compound of any one of claims 1-6, wherein R⁴ is selected from hydrogen, C₁-C₄ alkyl and O—C₁-C₄ alkyl, wherein each alkyl portion of R⁴ is substituted with 0-3 occurrences of R^(a).
 8. The compound of claim 7, wherein R⁴ is selected from hydrogen, —CH₃, and —OCH₃.
 9. The compound of any one of claims 1-8, wherein R⁵ is selected from hydrogen, C₁-C₄ alkyl substituted with 0-3 occurrences of R^(a).
 10. The compound of claim 9, wherein R⁵ is selected from hydrogen and —CH₃.
 11. The compound of any one of claims 1-10, wherein each R⁶ is independently selected from hydrogen, halo, and C₁-C₄ alkyl substituted with 0-3 occurrences of R^(a).
 12. The compound of claim 11, wherein each R⁶ independently selected from hydrogen, chloro and —CH₃.
 13. The compound of claim 12, wherein no more than one R⁶ is other than hydrogen.
 14. The compound of any one of claims 1-13, wherein ring A is a 6-membered monocyclic heteroaryl comprising at least one nitrogen ring atom.
 15. The compound of claim 14, wherein ring A is selected from


16. The compound of any one of claims 1-15, wherein n is 1; and R⁷ is pyrazol-1-yl substituted with 0-3 occurrences of R^(b).
 17. The compound of claim 16, wherein R⁷ is selected from 4-fluoropyrazol-1-yl and pyrazol-1-yl.
 18. The compound of any one of claims 1-17, wherein R⁹ is selected from hydrogen and C₁-C₄ alkyl substituted with 0-3 occurrences of R^(a).
 19. The compound of claim 18, wherein R⁹ is selected from hydrogen and methyl.
 20. The compound of claim 1 having the structural Formula (Ia):

or a pharmaceutically acceptable salt thereof, wherein ring A, X¹, X², R¹, R², R^(3a), R^(3b), R⁴, R⁵, R⁶, R⁷, R^(8a), R^(8b), R⁹, m and n are as defined as for structural Formula (I).
 21. The compound of claim 1 having the structural Formula (Ib):

or a pharmaceutically acceptable salt thereof, wherein ring A, X¹, X², R¹, R², R^(3a), R^(3b), R⁴, R⁵, R⁶, R⁷, R^(8a), R^(8b), R⁹, m and n are as defined as for structural Formula (I).
 22. A compound having the structural Formula (II):

or a pharmaceutically acceptable salt thereof, wherein: X¹ is selected from N, CH and C(halo); X² is selected from N and CH; X³ is selected from N and CH; R¹² is selected from hydrogen, hydroxyl, halo and optionally substituted O—C₁-C₄ alkyl; R¹⁴ is selected from hydrogen, optionally substituted —C₁-C₄ alkyl and optionally substituted —O—C₁-C₄ alkyl; R¹⁵ is selected from hydrogen and optionally substituted —C₁-C₄ alkyl; R¹⁶ is selected from hydrogen and optionally substituted —C₁-C₄ alkyl; R¹⁷ is selected from hydrogen and halo; and R¹⁹ is selected from hydrogen and optionally substituted —C₁-C₄ alkyl.
 23. The compound of claim 22, wherein: X¹ is selected from N, CH and C(Cl); X² is selected from N and CH; X³ is selected from N and CH; R¹² is selected from hydrogen, hydroxyl, fluoro and —O—CH₃; R¹⁴ is selected from hydrogen, —CH₃, and —OCH₃; R¹⁵ is selected from hydrogen and —CH₃; R¹⁶ is selected from hydrogen and —CH₃; R¹⁷ is selected from hydrogen and fluoro; and R¹⁹ is selected from hydrogen and —CH₃.
 24. The compound of claim 23, wherein when X¹ is CH, R¹⁶ is —CH₃.
 25. The compound of claim 22 having the structural Formula (IIa):

or a pharmaceutically acceptable salt thereof, wherein X¹, X², X³, R¹², R¹⁴, R¹⁵, R¹⁶, R^(17a), R^(17b), R^(17c), R^(18a), R^(18b) and R¹⁹ are as defined as for structural Formula (II).
 26. The compound of claim 22, having the structural Formula (IIb):

or a pharmaceutically acceptable salt thereof, wherein X¹, X², X³, R¹², R^(13a), R^(13b), R¹⁴, R¹⁵, R¹⁶, R^(17a), R^(17b), R^(17c), R^(18a), R^(18b) and R¹⁹ are as defined as for structural Formula (II).
 27. A pharmaceutical composition comprising a compound of any one of claims 1 to 26; and a pharmaceutically acceptable carrier.
 28. A method for inhibiting RET activity in a cell or in a patient, comprising the step of contacting the cell or administering to the patient a compound of any one of claims 1 to 26 or a pharmaceutical composition of claim
 27. 29. A method for treating a subject suffering from a condition mediated by aberrant RET activity, comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 26 or a pharmaceutical composition of claim
 27. 30. A method for treating a subject who has developed resistance to a cancer treatment, comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 26 or a pharmaceutical composition of claim
 27. 31. The method of claim 30, wherein the subject has developed resistance to a wild-type RET inhibitor.
 32. A method of treating cancer in a subject, said method comprising: a. determining if, having determined if, or receiving information that the subject has a RET-altered cell, cancer, gene, or gene product, e.g., has a fusion or point mutation; b. identifying the patient as responsive to a compound of any of claims 1-26 when the subject has the RET-altered cell, cancer, gene, or gene product; and c. administering an effective amount of the compound to the subject. 